Methods and materials for treating and preventing inflammation of mucosal tissue

ABSTRACT

The invention involves methods and materials for treating and preventing non-invasive fungus-induced mucositis. Specifically, the invention involves administrating an antifungal agent such that it contact mucus in an amount, at a frequency, and for a duration effective to prevent, reduce, or eliminate non-invasive fungus-induced rhinosinusitis. This invention also provides methods and materials for diagnosing non-invasive fungus-induced rhinosinusitis and culturing non-invasive fungus from a mammalian mucus sample as well as specific antifungal formulations and medical devices for treating and preventing non-invasive fungus-induced rhinosinusitis. In addition, the invention provides methods and materials for treating and preventing other non-invasive fungus-induced mucositis conditions such as chronic otitis media, chronic colitis, and Crohn&#39;s disease. Further, the invention involves methods and materials for treating and preventing chronic asthma symptoms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/062,709, filed Oct. 22, 1997, U.S. Provisional Application Ser.No. 60/063,414, filed Oct. 28, 1997, U.S. Provisional Application Ser.No. 60/063,418, filed Oct. 28, 1997, U.S. Provisional Application Ser.No. 60/083,272, filed Apr. 28, 1998 and U.S. Provisional ApplicationSer. No. 60/086,397, filed May 22, 1998.

BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in the treatmentand prevention of non-invasive fungus-induced inflammation of mucosaltissue as well as asthma symptoms.

2. Background Information

Mucositis, the inflammation of mucosal tissue, is a serious medicalproblem that affects millions of people worldwide. For example,conservative estimates indicate that between 20 to 40 million Americanssuffer from chronic rhinosinusitis, an inflammation of the nasal cavityand/or paranasal sinuses.

For the most part, the cause of chronic rhinosinusitis is unknown. In asmall percentage of patients, however, non-invasive fungal organismsliving within mucus seem to be involved. Patients having this condition,now known as allergic fungal sinusitis (AFS), were first described inthe early 1980's (Miller J W et al., Prod. Scot. Thor. Soc. 36:710(1981) and Katzenstein A L A et al., J. Allergy Clin. Immunol. 72:89-93(1983)). Specifically, about three to eight percent of chronicrhinosinusitis cases requiring surgery because of nasal obstructioncaused by polyp formation have been classified as AFS. Briefly, AFS isdiagnosed by the presence of inspissated mucus in the nasal-paranasalcavities. Typically, this mucus contains clumps or sheets of necroticeosinophils, Charcot-Leyden crystals, and non-invasive fungal hyphae. Inaddition, patients with AFS typically have a history of nasal-paranasalpolyposis and may have undergone multiple surgeries. Inflammation canaffect all nasal-paranasal cavities, but also can be asymmetricinvolving only one side. Computed topography (CT) scans of patients withAFS have a characteristic appearance and often reveal bone erosion inadjacent structures. Indeed, destruction of bones adjacent to thesinuses and nasal areas ranging from 19 percent to 80 percent has beenreported.

Although fungal organisms seem to be the causative agent of AFS,successful treatment remains lacking. Currently, AFS patients as well asmost chronic rhinosinusitis patients receive surgical treatment with orwithout steroid therapy. Surgery helps clear the nasal-paranasalcavities when obstructed by polyps and steroid therapy helps controlinflammatory responses that seem to be causing tissue and bonedestruction. Unfortunately, patients treated with surgery alone almostalways experience recurrent rhinosinusitis symptoms and additional polypgrowth. In addition, prolonged use of steroids is associated withsignificant side effects and steroid therapy removal also leads torecurrent episodes of rhinosinusitis. For these reasons, peoplesuffering from chronic rhinosinusitis conditions typically experiencerepeated cycles of intense inflammation, surgery, and steroid therapyfollowed by recurrent intense inflammation. Thus, neither surgery norsteroid therapy is particularly effective or desirable as a long-termtreatment for chronic rhinosinusitis conditions.

SUMMARY

The present invention relates generally to methods and materials fortreating and preventing non-invasive fungus-induced mucositis. The term“mucositis” as used herein means an inflammation, as opposed to aninfection, of a mucus membrane. This invention is based on the discoverythat the condition known as AFS can be treated successfully by using anantifungal agent in an amount, at a frequency, and for a durationeffective to reduce inflammation caused by the presence of fungalorganisms within nasal-paranasal mucus. In addition, this invention isbased on the discovery that using an antifungal agent in an amount, at afrequency, and for a duration effective to maintain a reduced level offungal organisms within nasal-paranasal mucus can prevent AFS symptoms.Specifically, the invention involves administering an antifungal agentto a mammal such that the antifungal agent contacts the mammal's mucusand reduces the presence of fungal organisms in mucus. In addition tobeing the only known method for successfully treating and preventingAFS, the use of an antifungal agent is particularly advantageous to apatient when compared to other currently available medical approaches toAFS such as surgical treatments and steroid therapies. Such medicalapproaches can have side effects, can be costly, and may be associatedwith patient discomfort.

This invention is also based on the discovery that most, if not all,chronic rhinosinusitis conditions have a fungal etiology and that most,if not all, cases of chronic rhinosinusitis can be treated by using anantifungal agent in an amount, at a frequency, and for a durationeffective to reduce the presence of fungal organisms withinnasal-paranasal mucus. In addition, using an antifungal agent in anamount, at a frequency, and for a duration effective to maintain areduced level of fungal organisms within nasal-paranasal mucus canprevent chronic rhinosinusitis symptoms.

This discovery is contrary to the current understanding of chronicrhinosinusitis and has far-reaching implications within medicine. Forexample, numerous medical research articles report that about three toeight percent of chronic rhinosinusitis cases requiring surgery are AFS,a rhinosinusitis condition having a non-invasive fungal etiology. Infact, less than 150 cases of AFS have been reported in the literatureover the past 15 years. It is noted that the lack of appreciation forthe non-invasive fungal etiology of chronic rhinosinusitis conditionsmay have occurred since affected individuals are frequently found tohave bacterial infections (i.e., invasive bacteria). Presumably, tissuedamage caused by non-invasive fungus-induced inflammation results in ahigher occurrence of bacterial infections in those damaged areas. Thus,overlaying bacterial infections in affected individuals could havemasked the underlaying cause, fungal organisms within mucus.

For the purpose of this invention, the term “non-invasive fungus-inducedrhinosinusitis” includes AFS as well as any other nasal-paranasalmucositis condition having a non-invasive fungal etiology.

Treating and preventing non-invasive fungus-induced rhinosinusitis,whether diagnosed as AFS or any other rhinosinusitis condition having anon-invasive fungal etiology, by using an antifungal agent circumventsthe need for surgical treatments and steroid therapies that causesignificant pain and suffering to the patient. Moreover, the use ofantifungal agents to treat and prevent non-invasive fungus-inducedrhinosinusitis actually directs treatment against the etiological agent(i.e., fungus), unlike surgical treatments, steroid therapies, andantibacterial treatments.

The term “chronic” as used herein refers to afflictions present for atleast three months. It is to be understood that afflictions that aretreated as described herein and become asymptomatic can be classified aschronic. Thus, chronic afflictions can be symptomatic or asymptomatic.

This invention is also based on another, equally significant, discoverythat chronic asthma symptoms can be treated and prevented successfullyby using an antifungal agent in an amount, at a frequency, and for aduration effective to reduce the presence of fungal organisms withinairway mucus. It is also apparent from the present discoveries thatantifungal agents can be administered directly to the lung airways forthe treatment of chronic asthma. Again, these discoveries are contraryto the current understanding of chronic asthma and have far-reachingclinical implications. Taken together, these significant breakthroughscan potentially allow large populations to experience happier,healthier, and more productive daily lives.

Specifically, the invention provides methods and materials for treatingand preventing a wide variety of mucoinflammatory diseases by using anantifungal agent. The use of an antifungal agent is a safe and highlyeffective treatment approach that involves mucoadministering anantifungal agent in an amount, at a frequency, and for a durationeffective to reduce, prevent, or eliminate a non-invasive fungus-inducedmucositis. The term “mucoadministration” as used herein refers to anytype of administration that places an administered agent in contact withmucus. This invention also provides specific antifungal formulationsthat can be applied to the various parts of a mammal that contain mucus.In addition, the invention provides medical devices that can be used toapply antifungal formulations. These devices are particularlyadvantageous since they can be used by an individual to administer aneffective dose of a specific antifungal formulation to the appropriatearea of the body. Further, the invention provides improved methods andmaterials for collecting and culturing fungal organisms from mucussamples. These culturing techniques can be used to monitor the number offungal species within mucus during a particular antifungal treatmentregimen. In addition, these fungus collecting and culturing methods andmaterials are useful for identifying the genotype and phenotype ofspecific fungal organisms that cause non-invasive fungus-inducedmucositis. The identification and characterization of non-invasivefungal organisms found in a particular individual's mucus can assistclinicians in determining proper treatment and prophylactic approaches.For example, this information can help determine the specific antifungalagent, amount, mode of administration, and number of applications to beused as well as possible combinatorial therapies that may include othermedications and procedures such as steroids, antibacterial agents, andsurgery.

In general, the invention features a method for treating a mammal (e.g.,human) having non-invasive fungus-induced rhinosinusitis. This methodinvolves directly mucoadministering to at least a portion of themammal's nasal-paranasal anatomy a formulation in an amount, at afrequency, and for a duration effective to reduce or eliminatenon-invasive fungus-induced rhinosinusitis. This formulation contains anantifungal agent or a plurality of antifungal agents and can be in asolid, liquid, or aerosol form (e.g., a powder, crystalline substance,gel, paste, ointment, salve, cream, solution, suspension, partialliquid, spray, nebulae, mist, atomized vapor, aerosol, and tincture). Inaddition, the formulation can be in a form suitable forself-mucoadministration by a human.

Further, the formulation can contain a pharmaceutically acceptableaqueous vehicle (e.g., saline and water). For example, a liquid form ofthe formulation can contain about 0.00001 percent to about 20 percent ofan antifungal agent as determined by antifungal agent weight per aqueousvehicle volume. In addition, the formulation can contain about 0.01 ngto about 1000 mg of an antifungal agent (e.g., amphotericin B) per literin some embodiments of the invention, or about 1 ng to about 500 mg ofan antifungal agent per liter in other embodiments of the invention, orabout 100 mg of an antifungal agent per liter in still other embodimentsof the invention. In addition, an effective amount of these aqueousformulations can be about 0.01 mL to about 1 L of formulation pernostril in some embodiments of the invention, or about 5 mL to about 100mL of formulation per nostril in other embodiments of the invention, orabout 40 mL of formulation per nostril in still other embodiments of theinvention. Alternatively, an effective amount of a formulation can beabout 0.01 ng to about 1000 mg of an antifungal agent per kg of bodyweight of the mammal in some embodiments of the invention or about 1 ngto about 500 mg of an antifungal agent per kg of body weight of themammal in other embodiments of the invention. The effective amount of aformulation can change or remain the same during an effective duration.The effective frequency of direct mucoadministration can be from aboutfour times a day to about once every other week in some embodiments ofthe invention, or from about twice a day to about once a week in otherembodiments of the invention, or about twice a day in still otherembodiments of the invention. In addition, the effective frequency ofdirect mucoadministration can be greater than once a day, or greaterthan once a week. The effective duration can be greater than about 7,14, 30, 60, or 90 days.

The mammal can be atopic or nonatopic and can be immunocompetent orimmunocompromised. In addition, the non-invasive fungus-inducedrhinosinusitis can be characterized by polyp formation or polypoidchange. The non-invasive fungus-induced rhinosinusitis also can be achronic condition. Mucoadministration can be an irrigation of at least aportion of the nasal-paranasal anatomy with a liquid form of theformulation. Alternatively, the mucoadministration can involve applyingan aerosol form of the formulation to at least a portion of thenasal-paranasal anatomy. An antifungal agent can be in a solid, liquid,or aerosol form. In addition, an antifungal agent can be a polyenemacrolide, tetraene macrolide, pentaenic macrolide, fluorinatedpyrimidine, imidazole, azole, triazole, halogenated phenolic ether,thiocarbamate, allylamine, sterol inhibitor, and an agent thatinterpolates fungal cell wall components. Such antifungal agents includeamphotericin B, flucytosine, ketoconazole, miconazole, itraconazole,fluconazole, griseofulvin, clotrimazole, econazole, terconazole,butoconazole, oxiconazole, sulconazole, saperconazole, voriconazole,ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafinehydrochloride, morpholines, nystatin, natamycin, butenafine, undecylenicacid, Whitefield's ointment, propionic acid, and caprylic acid. Inaddition to containing an antifungal agent, the formulation can contain,without limitation, a pharmaceutically acceptable aqueous vehicle,pharmaceutically acceptable solid vehicle, steroid, mucolytic agent,antibacterial agent, anti-inflammatory agent, immunosuppressant,dilator, vaso-constrictor, decongestant, leukotriene inhibitor,anti-cholinergic, anti-histamine, therapeutic compound, and combinationsthereof.

The method can also involve administering a second formulation thatcontains, without limitation, an antifungal agent, antibacterial agent,steroid, mucolytic agent, anti-inflammatory agent, immunosuppressant,dilator, vaso-constrictor, decongestant, leukotriene inhibitor,anti-cholinergic, anti-histamine, therapeutic compound, or combinationthereof. Likewise, the method can involve an additional step after thedirect mucoadministration. This additional step can be a prophylacticmucoadministration of a prophylactic formulation to the mammal in anamount, at a frequency, and for a duration effective to preventnon-invasive fungus-induced rhinosinusitis. This prophylacticformulation also contains an antifungal agent and can be in a solid,liquid, or aerosol form (e.g., powder, crystalline substance, gel,paste, ointment, salve, cream, solution, suspension, partial liquid,spray, nebulae, mist, atomized vapor, aerosol, tincture, pill, capsule,tablet, and gelcap). In addition, the prophylactic mucoadministrationcan be a direct or indirect mucoadministration. For example, theprophylactic mucoadministration can be an irrigation of at least aportion of the nasal-paranasal anatomy with a liquid form of theprophylactic formulation, an application of an aerosol form of theprophylactic formulation to at least a portion of the nasal-paranasalanatomy, or an oral administration of the prophylactic formulation tothe mammal in the form of a solid or liquid.

It will be understood that each of the additional features of theinvention described above can be applied to the following additionalembodiments and aspects of the invention. For example, methods forprophylactically treating a mammal at risk for developing non-invasivefungus-induced rhinosinusitis, and methods for treating asthma, mayutilize a formulation in which the antifungal agent is about 0.00001percent to about 20 percent by weight or volume of a formulation, and soforth.

In another embodiment, the invention features a method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced rhinosinusitis. This method involves mucoadministering tothe mammal a formulation in an amount, at a frequency, and for aduration effective to prevent non-invasive fungus-inducedrhinosinusitis. This formulation contains an antifungal agent.

Another embodiment of the invention features a method for treating amammal having non-invasive fungus-induced rhinosinusitis. This methodinvolves the steps of identifying (e.g., diagnosing) the mammal, anddirectly mucoadministering a formulation to at least a portion of thenasal-paranasal anatomy of the mammal in an amount, at a frequency, andfor a duration effective to reduce or eliminate non-invasivefungus-induced rhinosinusitis. This formulation contains an antifungalagent.

Another embodiment of the invention features a method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced rhinosinusitis. This method involves the steps ofidentifying the mammal (e.g., diagnosing), and mucoadministering aformulation to at least a portion of the nasal-paranasal anatomy of themammal in an amount, at a frequency, and for a duration effective toprevent non-invasive fungus-induced rhinosinusitis. This formulationcontains an antifungal agent.

In another aspect, the invention features a method for treating a mammalhaving asthma. This method involves directly mucoadministering to atleast a portion of the airways (e.g., nasal-paranasal airways and lungairways) of the mammal a formulation in an amount, at a frequency, andfor a duration effective to reduce or eliminate asthma symptoms. Thisformulation contains an antifungal agent. The direct mucoadministrationcan be the irrigation of the nasal-paranasal anatomy of the mammal witha liquid form of the formulation. Alternatively, the directmucoadministration can be the inhalation of the formulation through themammal's mouth or nose. In addition, the method can involve anadditional step after the direct mucoadministration. This additionalstep can be the prophylactic mucoadministration of a prophylacticformulation to the mammal in an amount, at a frequency, and for aduration effective to prevent asthma symptoms. This prophylacticformulation also contains an antifungal agent.

In another embodiment, the invention features a method forprophylactically treating a mammal at risk for developing asthma. Thismethod involves mucoadministering to at least a portion of the airways(e.g., nasal-paranasal airways and lung airways) of the mammal aformulation in an amount, at a frequency, and for a duration effectiveto prevent asthma symptoms. This formulation contains an antifungalagent.

Another embodiment of the invention features a method for treating amammal having asthma. This method involves the steps of identifying(e.g., diagnosing) the mammal, and directly mucoadministering aformulation to at least a portion of the airways (e.g., nasal-paranasalairways and lung airways) of the mammal in an amount, at a frequency,and for a duration effective to reduce or eliminate asthma symptoms.This formulation contains an antifungal agent.

Another embodiment of the invention features a method forprophylactically treating a mammal at risk for developing asthma. Thismethod involves the steps of identifying the mammal (e.g., diagnosing),and mucoadministering a formulation to at least a portion of the airways(e.g., nasal-paranasal airways and lung airways) of the mammal in anamount, at a frequency, and for a duration effective to prevent asthmasymptoms. This formulation contains an antifungal agent.

In another aspect, the invention features a method for treating a mammalhaving non-invasive fungus-induced intestinal mucositis (e.g., chroniccolitis and Crohn's disease). This method involves mucoadministering tothe mammal a formulation in an amount, at a frequency, and for aduration effective to reduce or eliminate non-invasive fungus-inducedintestinal mucositis symptoms. This formulation contains an antifungalagent and can be in the form of a regulated release capsule (e.g., pH ortime regulated release capsule). The mucoadministration can be the oralapplication of the formulation within the digestive tract of the mammal.Alternatively, the mucoadministration can be the application of theformulation within the digestive tract of the mammal by way of an enema.

In another embodiment, the invention features a method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced intestinal mucositis (e.g., chronic colitis and Crohn'sdisease). This method involves mucoadministering to the mammal aformulation in an amount, at a frequency, and for a duration effectiveto prevent non-invasive fungus-induced intestinal mucositis symptoms.This formulation contains an antifungal agent.

In another aspect, the invention features a method for treating a mammalhaving non-invasive fungus-induced otitis media. This method involvesmucoadministering to the mammal a formulation in an amount, at afrequency, and for a duration effective to reduce or eliminatenon-invasive fungus-induced otitis media. This formulation contains anantifungal agent. The mucoadministration can be the application of theformulation within the middle ear of the mammal. For example, a liquidform of the formulation can be used to irrigate the middle ear if thetympanic membrane is elevated or not intact. Alternatively, aformulation can be injected into the middle ear or a myringotomy can beused to penetrate the tympanic membrane. In addition, myringotomy tubescan be used to bypass the tympanic membrane. Further, a formulation canbe mucoadministered to the middle ear through the nose and eustachiantube.

In another embodiment, the invention features a method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced otitis media. This method involves mucoadministering tothe mammal a formulation in an amount, at a frequency, and for aduration effective to prevent non-invasive fungus-induced otitis media.This formulation contains an antifungal agent.

In another aspect, the invention features an article of manufacture thatcontains packaging material (e.g., boxes, wrappings, vials, and othercontainers) and a formulation contained within the packaging material.This formulation contains an antifungal agent. The packaging materialcontains a label or package insert indicating that the formulation canbe directly mucoadministered to at least a portion of thenasal-paranasal anatomy of a mammal having non-invasive fungus-inducedrhinosinusitis in an amount, at a frequency, and for a durationeffective to reduce or eliminate non-invasive fungus-inducedrhinosinusitis.

In another embodiment, the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent andthe packaging material contains a label or package insert indicatingthat the formulation can be mucoadministered to at least a portion ofthe nasal-paranasal anatomy of a mammal at risk for developingnon-invasive fungus-induced rhinosinusitis in an amount, at a frequency,and for a duration effective to prevent non-invasive fungus-inducedrhinosinusitis.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent. Thepackaging material contains a label or package insert indicating thatthe formulation can be directly mucoadministered to at least a portionof the airways of a mammal having asthma in an amount, at a frequency,and for a duration effective to reduce or eliminate asthma symptoms.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent andthe packaging material contains a label or package insert indicatingthat the formulation can be mucoadministered to at least a portion ofthe airways of a mammal at risk for developing asthma in an amount, at afrequency, and for a duration effective to prevent asthma symptoms.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent. Thepackaging material contains a label or package insert indicating thatthe formulation can be mucoadministered to a mammal having non-invasivefungus-induced intestinal mucositis in an amount, at a frequency, andfor a duration effective to reduce or eliminate non-invasivefungus-induced intestinal mucositis symptoms.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent andthe packaging material contains a label or package insert indicatingthat the formulation can be mucoadministered to a mammal at risk fordeveloping non-invasive fungus-induced intestinal mucositis in anamount, at a frequency, and for a duration effective to preventnon-invasive fungus-induced intestinal mucositis symptoms.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent. Thepackaging material contains a label or package insert indicating thatthe formulation can be mucoadministered to a mammal having non-invasivefungus-induced otitis media in an amount, at a frequency, and for aduration effective to reduce or eliminate non-invasive fungus-inducedotitis media symptoms.

Another embodiment of the invention features an article of manufacturethat contains packaging material and a formulation contained within thepackaging material. This formulation contains an antifungal agent andthe packaging material contains a label or package insert indicatingthat the formulation can be mucoadministered to a mammal at risk fordeveloping non-invasive fungus-induced otitis media in an amount, at afrequency, and for a duration effective to prevent non-invasivefungus-induced otitis media symptoms.

In another aspect, the invention features the use of an antifungal agentfor the manufacture of a medicament for the treatment or prevention ofnon-invasive fungus-induced rhinosinusitis.

In another embodiment, the invention features the use of an antifungalagent for the manufacture of a medicament for the treatment orprevention of asthma symptoms.

Another embodiment of the invention features the use of an antifungalagent for the manufacture of a medicament for the treatment orprevention of non-invasive fungus-induced intestinal mucositis.

Another embodiment of the invention features the use of an antifungalagent for the manufacture of a medicament for the treatment orprevention of non-invasive fungus-induced otitis media.

In another aspect, the invention features an antifungal formulationcontaining an antifungal agent, a flavoring, and water. The waterconstitutes at least about 50 percent of the formulation. For example,the water can constitute at least about 55, 60, 65, 70, 75, 80, 85, 90,95, or 99 percent of the formulation.

In another embodiment, the invention features an antifungal formulationcontaining itraconazole and water. The itraconazole is dissolved in theformulation at a concentration greater than about 25 μg per mL. Forexample, the itraconazole can be dissolved in the formulation at aconcentration greater than about 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, or 180 μg per mL. In addition, the waterconstitutes at least about 50 percent of the formulation. For example,the water can constitute at least about 55, 60, 65, 70, 75, 80, 85, 90,95, or 99 percent of the formulation. The formulation also can containpolyethylene glycol (e.g., PEG-200, PEG400, PEG-800, etc.). Theformulation also can contain flavoring (e.g., peppermint oil, cherryflavoring, syrup, and the like).

In another embodiment, the invention features an antifungal formulationcontaining itraconazole and water. The itraconazole is suspended in theformulation at a concentration greater than about 25 μg per mL. Forexample, the itraconazole can be suspended in the formulation at aconcentration greater than about 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, or 180 μg per mL. In addition, the waterconstitutes at least about 50 percent of the formulation. For example,the water can constitute at least about 55, 60, 65, 70, 75, 80, 85, 90,95, or 99 percent of the formulation. The formulation also can containpolyethylene glycol (e.g., PEG-200, PEG-400, PEG-800, etc.). Theformulation also can contain flavoring (e.g., peppermint oil, cherryflavoring, syrup, and the like).

In another embodiment, the invention features an antifungal formulationcontaining an antifungal agent, a flavoring, and water. The waterconstitutes at least about 50 percent of the formulation. For example,the water can constitute at least about 55, 60, 65, 70, 75, 80, 85, 90,95, or 99 percent of the formulation. In addition, the antifungal agentcan be amphotericin B, ketoconazole, saperconazole, voriconazole,flucytosine, miconazole, fluconazole, griseofulvin, clotrimazole,econazole, terconazole, butoconazole, oxiconazole, sulconazole,ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafinehydrochloride, morpholines, nystatin, natamycin, butenafine, undecylenicacid, Whitefield's ointment, propionic acid, and caprylic acid.

In another aspect, the invention features a method of making anantifungal formulation. The formulation contains itraconazole and water.The itraconazole is dissolved in the formulation at a concentrationgreater than about 25 μg per mL. For example, the itraconazole can bedissolved in the formulation at a concentration greater than about 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180μg per mL. The water constitutes at least about 50 percent of theformulation. For example, the water can constitute at least about 55,60, 65, 70, 75, 80, 85, 90, 95, or 99 percent of the formulation. Themethod includes adding the water to a stock solution containing theitraconazole.

In another aspect, the invention features a method for culturing fungusfrom a mammal's mucus. The method includes (1) contacting the mucus witha mucolytic agent to reduce the viscosity of the mucus, (2) separatingthe fungus from the reduced-viscosity mucus, (3) contacting theseparated fungus with fungus growth medium to form a fungus culture, and(4) incubating the fungus culture such that the separated fungus grows.

In another aspect, the invention features a method for obtaining afungal antigen. The method includes (1) contacting a mammal's mucus witha mucolytic agent to reduce the viscosity of the mucus, (2) separatingfungus from the reduced-viscosity mucus, (3) contacting the separatedfungus with fungus growth medium to form a fungus culture, (4)incubating the fungus culture such that the separated fungus grows, and(5) isolating the antigen from the cultured fungus.

In another aspect, the invention features a method for producing afungus-specific antibody. The method includes (1) contacting a mammal'smucus with a mucolytic agent to reduce the viscosity of the mucus, (2)separating fungus from the reduced-viscosity mucus, (3) contacting theseparated fungus with fungus growth medium to form a fungus culture, (4)incubating the fungus culture such that the separated fungus grows, (5)isolating a fungal antigen from the cultured fungus, and (6) immunizingan animal with the fungal antigen to produce the antibody.

In another aspect, the invention features a nasal mucus collectingapparatus. The apparatus contains a collection retainer, a collectiontube, and a connecting portion. The collection retainer is suitable forretaining mucus. The collection tube extends from the collectionretainer and defines a distal end and a lumen such that mucus cantraverse the lumen from the distal end of the collection tube to thecollection retainer. The collection tube is generally flexible over atleast a portion of the tube's length such that the collection tube canbe selectively manipulated into a desired configuration by apractitioner during a collection procedure. The collection tube furtheris generally malleable such that the collection tube generally retainsthe desired configuration until the practitioner manipulates thecollection tube to conform to a different configuration. The connectingportion extends from the collection retainer and defines a second lumenthat communicates with the interior of the collection retainer. Theconnecting portion is adapted to receive a vacuum source. In addition,the apparatus can further contain a valve that adjusts the opening ofthe second lumen. The collection retainer can be removable from thecollection tube and the connection portion.

In another aspect, the invention features a pharmaceutical compositioncontaining an antifungal agent.

In another embodiment, the invention features a pharmaceuticalcomposition containing an antifungal agent and an mucolytic agent.

Another embodiment features a pharmaceutical composition containing anantifungal agent and a steroid.

Another embodiment features a pharmaceutical composition containing anantifungal agent and a decongestant.

Another embodiment features a pharmaceutical composition containing anantifungal agent and an antibiotic.

Another embodiment features a pharmaceutical composition containing anantifungal agent and an anti-inflammatory.

Another embodiment features a pharmaceutical composition containing anantifungal agent and an anti-histamine.

Another embodiment features a pharmaceutical composition containing anantifungal agent and an anti-cholinergic.

Another embodiment features a pharmaceutical composition containing anantifungal agent and a leukotriene inhibitor.

In another aspect, the invention features a composition for treating animmune response to fungus in a mammal, characterized by an agentconfigured for direct mucoadministration to the mucus of the mammal andhaving antifungal means for eliminating or reducing the fungus below athreshold level wherein the fungus ceases to activate eosinophilemigration to the affected area.

In another aspect, the invention features a pharmaceutical compositionfor treating a fungal related condition in the nasal-sinus anatomy,pulmonary anatomy, ear anatomy, or intestinal anatomy of a mammalianpatient, said composition comprising an effective dose of an anti-fungalas described herein.

In another aspect, the invention features a pharmaceutical compositionfor treating a fungal related condition in the nasal-sinus anatomy,pulmonary anatomy, ear anatomy, or intestinal anatomy of a mammalianpatient, said composition comprising an effective dose of an anti-fungaland at least one other agent or inhibitor as described herein.

In another aspect, the invention features a pharmaceutical compositionfor treating a fungal related condition in the nasal sinus anatomy,pulmonary anatomy, ear anatomy, or intestinal anatomy of a mammalianpatient, said composition comprising an effective dose of an anti-fungalsuitable for long term use within the nasal-sinus anatomy.

In another aspect, the invention features a medication for treatingsinusitis, asthma, otitis media, or colitis of a patient, comprising amucolytic agent; and an anti-fungal compound as described herein.

In another aspect, the invention features an irrigation medication fortreating an inflamed nasal area, lung area, ear area, or intestinal areaof a patient, the inflamed nasal area, lung area, ear area, orintestinal area being caused by the presence of a fungus, the medicationcomprising effective doses of an antifungal compound and a steroid asdescribed herein.

In another aspect, the invention features an irrigation medication fortreating an inflamed nasal area, lung area, ear area, or intestinal areaof a patient, the inflamed nasal area, lung area, ear area, orintestinal area being caused by the presence of a fungus, the medicationcomprising effective doses of an antifungal compound and a mucolyticagent.

In another aspect, the invention features an irrigation medication fortreating an inflamed nasal area, lung area, ear area, or intestinal areaof a patient, the inflamed nasal area, lung area, ear area, orintestinal area being caused by the presence of a fungus, the medicationcomprising effective doses of a steroid and a mucolytic agent asdescribed herein.

In another aspect, the invention features an irrigation medication fortreating an inflamed nasal area, lung area, ear area, or intestinal areaof a patient, the inflamed nasal area, lung area, ear area, orintestinal area being caused by the presence of a fungus, the medicationcomprising effective doses of an antifungal compound, a steroid, and amucolytic agent as described herein.

In another aspect, the invention features an irrigation medication fortreating an inflamed nasal area, lung area, ear area, or intestinal areaof a patient, the inflamed nasal area, lung area, ear area, orintestinal area being caused by the presence of a fungus, the medicationcomprising an effective dose of at least one medicine selected from thegroup consisting of an antifungal compound, a steroid, a mucolyticagent, and any combination thereof as described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a CT scan of a patient with bilateral chronic rhinosinusitis.

FIG. 2 is a CT scan of the patient of FIG. 1, four months later, aftertreatment with antifungal irrigations.

FIG. 3 is a diagram depicting a device for collecting mucus.

FIG. 4 is a diagram depicting a device for collecting mucus.

DETAILED DESCRIPTION

The invention involves methods and materials for treating and preventingnon-invasive fungus-induced mucositis. Specifically, the inventioninvolves mucoadministering an antifungal agent in an amount, at afrequency, and for a duration effective to prevent, reduce, or eliminatechronic non-invasive fungus-induced rhinosinusitis. This invention alsoprovides methods and materials for diagnosing chronic non-invasivefungus-induced rhinosinusitis and culturing non-invasive fungus from amammalian mucus sample as well as specific antifungal formulations andmedical devices for treating and preventing non-invasive fungus-inducedrhinosinusitis. In addition, the invention provides methods andmaterials for treating and preventing other non-invasive fungus-inducedmucositis conditions such as chronic otitis media, chronic colitis, andCrohn's disease. Further, the invention involves methods and materialsfor treating and preventing chronic asthma symptoms.

Although not limited to any particular mode of action, the presentinvention involving the treatment and prevention of non-invasivefungus-induced inflammation of mucosal tissue by using an antifungalagent is based on the following proposed mechanism of diseaseprogression derived from the discoveries reported herein. In general,most, if not all, individuals have fungal organisms living in theirmucus. Normally, most individuals tolerate these non-invasive organismsand live normal disease-free lives. For unknown reasons, someindividuals do not tolerate these fungal organisms and begin to mount animmune response against them. As the immune response progresses,eosinophils accumulate within the local tissue. This accumulation ofeosinophils can contribute to the formation of obstructive tissue masses(e.g, polyps and polypoid structures) as well as the transmigration ofactivated eosinophils from the tissue (inside the body) to the mucus(outside the body). These obstructive tissue masses appear to preventnormal cavity clearance and thus can facilitate additional fungalgrowth. Once eosinophils are within the mucus, they can release thecontents of their granules presumably upon the activation of surface Fcreceptors. Eosinophil granules contain many toxic molecules such aseosinophil cationic protein (ECP), eosinophil peroxidase (EPO), andmajor basic protein (MBP). Upon release, these toxic molecules candamage both the targeted foreign microorganisms (e.g., fungus) as wellas self tissues. The degree of damage caused by eosinophil accumulationand eosinophil degranulation varies significantly from slightinflammatory pain and discomfort to major structural abnormalities suchas tissue and bone destruction and the formation of polyps, polypoidstructures, and other tumors. Once self tissues are damaged, theindividual can have an increased susceptibility to bacterial infectionsas well. Thus, the characteristic inflammatory responses, resultingdamages, and resulting bacterial infections observed within most, if notall, chronic rhinosinusitis patients are actually triggered bynon-invasive fungal organisms.

It is noted that fungal organisms may be observed within the tissueunder extreme mucositis conditions of tissue and bone destruction simplybecause the barrier (i.e., epithelium) between the inside and outside ofthe body has been destroyed or damaged. In these situations, the mereobserved presence of a small number of fungal organisms within alocalized area of tissue damage does not deter from the fact that theaffliction is a non-invasive fungus-induced mucositis and not aninfection.

The discovery that most, if not all, chronic rhinosinusitis cases arecaused by non-invasive fungal organisms suggests that other chronicinflammatory conditions such as chronic otitis media, chronic colitis,and Crohn's disease are most likely caused by non-invasive fungalorganisms living within mucus. In addition, the discovery thatnon-invasive fungus-induced rhinosinusitis can be successfully treatedand prevented with antifungal agents when mucoadministered in aneffective amount, at an effective frequency, and for an effectiveduration suggests that these other non-invasive fungus-induced mucositisconditions also can be treated and prevented with antifungal agents whenused appropriately as described herein. Further, the discovery thatchronic asthma symptoms can be treated and prevented with antifungalagents when directly mucoadministered to the airways by way ofnasal-paranasal irrigation suggests that antifungal agents can beeffective when directly mucoadministered to the airways by inhalationthrough the nose or mouth. Thus, the methods and materials describedherein have the potential to treat millions of people suffering fromchronic rhinosinusitis, chronic otitis media, chronic colitis, Crohn'sdisease, and any other non-invasive fungus-induced mucositis conditionas well as chronic asthma.

As described above, a non-invasive fungus-induced mucositis is aninflammation, not an infection. In general, inflammations arefundamentally and clinically different from infections. An infection isdefined as the growth of an organism within tissue. In addition, aninfection is characterized as an invasive disease meaning that aninfectious organism enters the tissue of a host and then triggers a hostimmune response and/or causes damage. Thus, the role of the infectiousorganism is typically that of an invasive pathogen. In addition, aninfected individual can be immunocompetent or immunocompromised. Whenthe infected individual is immunocompromised, the infection is oftentermed an “opportunistic” infection. Further, infections can be acute orchronic depending upon multiple factors such as the competence of theinfected individual's immune system, the nature of the invasivepathogen, and the availability of medical treatments.

In contrast, an inflammation is characterized as a localized protectiveresponse that serves to destroy, dilute, and/or sequester an injuriousagent or insult. In addition, inflammatory responses typically result inredness, swelling, heat, and pain. In the case of non-invasivefungus-induced mucositis, the localized protective response is against anon-invasive fungal organism living outside the tissue (e.g., withinmucus). Typically, some individuals suffering from a non-invasivefungus-induced mucositis are atopic and/or immunocompetent. In addition,the role of the injurious agent (i.e., fungus) is that of a non-invasiveallergen. Thus, a non-invasive fungus-induced mucositis is an allergicreaction mounted by the individual's immune system against a fungalorganism living outside an individual's tissue.

As described herein, the invention provides methods and materials thatreduce the presence of fungal organisms within mucus to a level and fora period of time such that the characteristic inflammatory responses andresulting damages associated with mucositis are stopped, treated, orprevented. For the purpose of clarity, reducing the presence of fungalorganisms within mucus to treat or prevent mucositis is similar toremoving an allergen (e.g., pollen) from the presence of an individualsuffering from an allergic reaction (e.g., hay fever). Again, theallergic reaction against, for example, pollen is not an infection butan inflammation. In addition, the simplicity of this inventionunderscores the far-reaching clinical implications that follow thesediscoveries. For example, once clinicians understand that most, if notall, chronic rhinosinusitis is caused by non-invasive fungal organismsand that this inflammatory disease can be treated and prevented byreducing the level of non-invasive fungal organisms withinnasal-paranasal mucus using the methods and materials described herein,then millions of people will be able to have healthier, happier, andmore productive lives.

Identifying Non-invasive Fungus-induced Mucositis

A non-invasive fungus-induced mucositis is defined as an inflammation ofany mucosal tissue induced by a non-invasive fungal organism. Examplesof mucosal tissue include, without limitation, the mucosa of the mouth,gut, nasal passages, paranasal sinuses, airways of the lung, trachea,middle ear, eustachian tube, vagina, and urethra. In general, aninflammation of a mucosal tissue can be determined using any of themethods commonly known to a skilled artisan. For example, an individualcan be identified as having an inflammation of a mucosal tissue uponexamination of a tissue biopsy as well as by visual examination,endoscopic analysis, and image analysis techniques (e.g., X-rays, CTscans, and magnetic resonance imagery (MRI) scans) since the variousinflamed mucosal anatomies tend to exhibit observable abnormalcharacteristics.

Multiple diagnostic methods can be used to determine if a particularmucositis is a non-invasive fungus-induced mucositis. In general, suchdiagnostic methods include, without limitation, reviewing the affectedindividual's prior medical conditions and treatments, interviewing andevaluating the affected individual, and collecting and analyzingbiological samples from the affected individual.

Reviewing an affected individual's medical history can be helpful indetermining if a particular mucositis is a non-invasive fungus-inducedmucositis since such inflammations are typically recurrent and chronic.Thus, signs of a previous non-invasive fungus-induced mucositis episodewould suggest that an instant mucositis is a non-invasive fungus-inducedmucositis as well. Other useful information within an individual'smedical history could include, without limitation, allergies, surgeries,and other diseases such as cystic fibrosis and ciliary dismotilitysyndromes.

Interviewing and evaluating an affected individual can also helpidentify a non-invasive fungus-induced mucositis. For example,individuals suffering from chronic mucositis symptoms such as airwayobstructions, loss of smell, loss of hearing, wheezing, dyspnea,coughing, head ache, and facial pressure may have a non-invasivefungus-induced mucositis. In addition, a non-invasive fungus-inducedmucositis can be ruled out in individuals that exhibit the symptoms ofan infection such as fever, fungal dissemination, fungemia, increase inpolymorphonuclear leukocytes, and acute onset. It is noted thatrecurrent bacterial infections can indicate an underlying non-invasivefungus-induced mucositis condition since chronic inflammation can leadto the destruction of the epithelium and thus increase the individual'ssusceptibility to bacterial infection. Further, multiple diagnostictests can be performed to help identify a non-invasive fungus-inducedmucositis. For example, a common allergy screen using a panel of fungaland non-fungal antigens can be used to determine if an individual isatopic since some cases of non-invasive fungus-induced mucositis involveatopic individuals. Further, immuno-based tests for the presence ofanti-fungal antigen antibodies, tests for abnormal pulmonary functionwith or without methacholine, audiograms, and tympanograms can be usedto identify a non-invasive fungus-induced mucositis.

Collecting and analyzing biological samples from an affected individualcan help identify a non-invasive fungus-induced mucositis. In general,biological samples such as mucus, stool, urine, sputum, and blood can becollected and analyzed for signs that indicate the involvement of anon-invasive fungal organism. Such signs can include, withoutlimitation, the presence of any antigenic marker for non-invasivefungus-induced mucositis; the presence of eosinophils, eosinophilproducts (e.g., MBP and ECP), antibodies, fungal antigens, or fungalorganisms within a mucus, stool, urine, or sputum sample; and theabsence of fungal organisms within a blood sample. For example, theidentification of allergic mucus (i.e., mucus that contains evidence ofeosinophil presence) can indicate a non-invasive fungus-inducedmucositis. Such evidence of eosinophil presence includes, withoutlimitation, the presence of intact eosinophils, necrotic eosinophils,and eosinophil products. Many methods for detecting the presence ofthese various signs and markers within a biological sample are wellknown in the art and can be used. For example, the presence ofeosinophils within allergic mucus can be determined using ahematoxylin/eosin stain followed by microscopic examination.

In addition, a tissue biopsy can be collected and analyzed for the lackof invasive fungus. As noted above, fungal organisms may be observedwithin tissue under extreme mucositis conditions of tissue and bonedestruction when examining a tissue biopsy simply because the barrier(i.e., epithelium) between the inside and outside of the body has beendestroyed or damaged. In these situations, the mere presence of a smallamount of fungal organisms within a localized area of tissue damage doesnot necessarily mean the affliction is not a non-invasive fungus-inducedmucositis.

Further, immuno-based assays can be used to detect the presence ofvarious signs of a non-invasive fungus-induced mucositis within abiological sample. Many immuno-based assays are well known in the artincluding, without limitation, enzyme-linked immunosorbent assays(ELISA) and radioallergosorbant tests (RAST). The methods of using RASTare described, for example, in McRury J et al. (Clin Exp Immunol65:631-638 (1986)), Mabry R L and Manning S (Otolaryngol Head Neck Surg.113:721-723 (1995), and Lynch N R et al. (Int Arch Allergy Immunol114:59-67 (1997)). Immuno-based assays can use polyclonal antibodies,monoclonal antibodies, or fragments thereof that have specificity for anantigen that can be used as a diagnostic marker for non-invasivefungus-induced mucositis. For example, monoclonal antibodies havingspecificity for fungal organisms known to cause non-invasivefungus-induced mucositis can be produced and used to screen biologicalsamples. Such antibodies can be produced using methods describedelsewhere (Zeidan et al., Experimental Approaches in Biochemistry andMolecular Biology, William C. Brown Publisher (1996) and Seaver,Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up,Marcel Dekker Inc., New York, N.Y. (1987)). Briefly, a mouse can beimmunized with a sample of a fungal organism isolate. Several weekslater lymphocytes from spleen of the immunized mouse can be recoveredand fused with myeloma cells to produce hybridoma cells. Hybridoma cellsexhibiting specificity for the immunizing fungal isolate then can beisolated and monoclonal antibody preparations produced.

Since the specific methods and materials used to identify non-invasivefungus-induced mucositis can vary depending upon the specific locationof the mucositis, a more detailed description is provided below forseveral exemplary mucosal tissues.

1. Nasal-paranasal Cavities

The external bony framework of the nose consists of two oblong nasalbones. One nasal bone is disposed on each side of a midline, with thetwo bones forming an arched cross section. The nasal septum divides thenasal cavity in half. The lateral nasal wall has three turbinates thatincrease the mucosal surface area of the nasal cavity or vestibule. Thenasal vestibule is bounded by the nasal septum and lateral wall. Thislarge surface area of the turbinates and nasal septum promotes extensivecontact with inspired air, thus facilitating humidification, particleremoval, and temperature regulation of inspired air.

The paranasal sinuses are air-containing spaces joining the nasal cavityby means of openings or ostia. Although they are paired, the paranasalsinuses are commonly asymmetrical in shape and location, and include themaxillary, frontal, ethmoid, and sphenoid sinuses. Suggested functionsof the paranasal sinuses include lightening the bones of the skull,providing mucus for the nasal cavity, and acting as resonant chambersfor the production of sound. The maxillary sinuses are the largest ofthe paranasal sinuses. Each maxillary sinus is located in the maxillaand opens into the middle meatus. The frontal sinuses are located in thefrontal bone and are superior and medial to the orbit of the eye. Thefrontal sinuses also empty into the middle meatus. The ethmoid sinusesare numerous and irregularly shaped air spaces opening into the middleand superior meatuses. The sphenoid sinus is in the sphenoid bone and isposterior to both the eye and the upper portion of the nasal cavity. Thesphenoid sinus drains into the superior meatus.

Mucosal tissue (mucosa) lines both the nasal cavity and the paranasalsinuses, and generally comprises an epithelial layer, connective tissue,and mucus glands. A layer of mucus normally covers the mucosa. Mucusthat is secreted from mucosa serves to trap particles and to preventdehydration of the nasal and paranasal tissues that are otherwiseexposed to air. The mucus is normally transported by cilia toward thenasopharynx and then swallowed.

Individuals suffering from rhinosinusitis can be identified usingmethods commonly known in the art. Symptoms of rhinosinusitis include,without limitation, nasal airway obstruction, loss of smell, facialpain, head ache, post nasal drip, and rhinorrhea. Upon examination, thepresence of thick mucus or the visual identification of nasal orparanasal obstruction with mucus or polyps often indicates arhinosinusitis condition. Nasal polyps are outgrowths from thenasal-paranasal mucosa that are typically smooth, gelatinous,semitranslucent, round or pear shaped, and pale. In general, nasalpolyps are located on the lateral wall of the nose, usually in themiddle meatus or along the middle and superior turbinates. Most nasalpolyps arise from the ethmoid sinus but some polyps originate in themaxillary sphenoid sinuses. The mass of a nasal polyp is composed mainlyof edematous fluid with sparse fibrous cells and a few mucous glands.The surface epithelium of nasal and paranasal polyps generally revealssquamous metaplasia. Eosinophils are usually present in polyps inmoderate to large numbers, and it is now known that nasal polyp fluidcontains greater than normal concentrations of IgA, IgE, IgG, and IgMantibodies as well as abnormally high concentrations of IL-5, a cytokinethat contributes to eosinophil activation and survival. As demonstratedherein, the presence of nasal polyps is not a risk factor forrhinosinusitis, but rather an end stage of chronic inflammation.

The following methods and materials can be used to identify individualssuffering from non-invasive fungus-induced rhinosinusitis. As describedabove, the condition known as AFS is a non-invasive fungus-inducedrhinosinusitis condition.

Thus, any method known in the art that is used to identify AFS can beused to identify non-invasive fungus-induced rhinosinusitis (Cody D T etal. Laryngoscope 104:1074-1079 (1994) and Kupferberg S B et al.,Otolaryngol. Head Neck Surg 117:35-41 (1997)). For example, non-invasivefungus-induced rhinosinusitis can be identified by the presence ofinspissated mucus that contains clumps or sheets of necroticeosinophils, Charcot-Leyden crystals, and non-invasive fungal hyphae. Inaddition, image analysis such as MRI and CT scans can be used toidentify non-invasive fungus-induced rhinosinusitis since suchconditions typically exhibit a characteristic appearance and often causebone erosion in adjacent structures (Quraishi et al., Otolaryngol. HeadNeck Surg. 117:29-34 (1997); Manning et al., Laryngoscope 107:170-176(1997); Kinsella et al., Head & Neck 18:211-217 (1996); Allbery et al.,RadioGraphics 15:1311-1327 (1995); Roth M R, Ear, Nose & Throat J.73:928-930 (1994); and Bartynski et al., Otolaryngol. Head Neck Surg.103:32-39 (1990)). Further, individuals with non-invasive fungus-inducedrhinosinusitis may have a history of nasal-paranasal polyposis and mayhave undergone multiple surgeries.

Results using currently available diagnostic methodology indicate thatabout three to eight percent of chronic rhinosinusitis cases requiringsurgery are AFS cases. In general, these current AFS diagnostic methodsinvolve criteria such as the presence of a characteristic appearance ona CT scan, the presence of allergic mucus, and the presence of fungalorganisms within mucus samples as confirmed by either histology orfungal growth in culture. The present invention demonstrates thatgreater than about 90 percent of all chronic rhinosinusitis cases have afungal etiology based on a better understanding of chronicrhinosinusitis, improved diagnostic procedures, and the impressivesuccess rate of the antifungal treatment approaches described herein. Inaddition, the present invention demonstrates that the ability to growfungal organisms from a mucus sample is not a useful criterion fordiagnosing a non-invasive fungus-induced rhinosinusitis condition, suchas AFS, since most, if not all, humans have fungal organisms withintheir nasal-paranasal mucus (See Example 1). It is noted, however, thatcollecting, analyzing, and/or culturing fungal organisms from anasal-paranasal mucus sample can provide useful diagnostic information.Such information can include, without limitation, information about thelevel of fungal organisms and the number of different fungal speciespresent within a particular mucus sample.

Lack of appreciation for the non-invasive fungal etiology of chronicrhinosinusitis conditions appears to have occurred for several reasons.First, reliance on inadequate mucus collecting and fungus culturingtechniques seems to have led to the misinterpretation of negative fungalgrowth results. As shown herein, these negative results were most likelyfalse-negative results since fungal organisms can be grown fromnasal-paranasal mucus samples collected from most, if not all, humans.Consequently, the ability to grow fungal organisms from anasal-paranasal mucus sample is essentially meaningless as a diagnosticcriterion for non-invasive fungus-induced rhinosinusitis conditionsincluding AFS. Second, clinicians, during surgery, routinely wash awayor discard mucus from the nasal-paranasal cavities prior to removing andexamining a polyp for the presence of allergic mucus. Thus, the failureto detect allergic mucus most likely resulted from a failure to collectthe proper medium in which to examine. This in turn may have led to thewidely recognized and medically accepted theory that polyposis is thecause of certain inflammatory conditions in the nasal-paranasalanatomies. As discussed above, polyposis can be considered an end-stageresult of chronic inflammation. Third, chronic inflammatory conditions,as described herein, can lead to recurrent bacterial infections that mayhave masked an underlying non-invasive fungus-induced rhinosinusitiscondition. In addition, any temporary relief observed afterantibacterial treatment may have complicated the diagnosis of acondition having a non-invasive fungal etiology.

Regardless, the present invention teaches that special care should betaken to preserve the mucus for analysis and that the presence ofallergic mucus can be used to identify non-invasive fungus-inducedrhinosinusitis. In addition, recurrent bacterial infections within thenasal-paranasal anatomy can indicate non-invasive fungus-inducedrhinosinusitis.

Any individual that had a previous episode of rhinosinusitis is at riskfor developing non-invasive fungus-induced rhinosinusitis. In addition,elderly individuals as well as individuals having cystic fibrosis,asthma, and a family history of nasal problems or allergies can be atrisk for developing non-invasive fungus-induced rhinosinusitis. Further,individuals that are exposed to significant levels of allergens (e.g.,fungus spores, pollen, and chemicals) can be at risk for developingnon-invasive fungus-induced rhinosinusitis.

2. Middle Ear

The ear can be divided into three parts: the external ear, middle ear,and internal ear. The middle ear is a cavity that is connected to thenasopharynx by the eustachian tube. In addition, the middle ear isseparated from the opening of the external ear by the tympanic membraneand contains a chain of three small bones that connect the tympanicmembrane with the internal ear. Mucosal tissue lines most of the middleear space.

Individuals suffering from an inflammation of the mucosal tissue of themiddle ear (otitis media) can be identified based on a medical historyof middle ear afflictions, visual examination, tissue biopsy, andsymptoms such as a loss of hearing, otorrhea, and middle ear effusion.

In general, a non-invasive fungus-induced otitis media condition can beidentified by (1) collecting and analyzing fluid or mucus samples fromthe middle ear for fungal organisms or eosinophil presence, (2) tissuebiopsy analysis for non-invasive fungal organisms, (3) an audiogramconsistent with conductive hearing loss, and (4) a flat tympanogram.Unlike nasal-paranasal mucus, identifying the presence of fungalorganisms in samples collected from the middle ear can indicate anon-invasive fungus-induced inflammatory condition since the middle earnormally is quite sterile.

Any individual that had a previous episode of otitis media is at riskfor developing non-invasive fungus-induced otitis media. In addition,young individuals (e.g., infants and toddlers) as well as individualshaving a family history of ear problems or allergies can be at risk fordeveloping non-invasive fungus-induced otitis media.

3. Intestines

Mucosal tissue lines both the small and large intestines. Individualssuffering from an inflammation of the intestines (e.g., ulcerativecolitis and Crohn's disease) can be identified using methods andmaterials commonly known in the art. For example, tissue biopsy analysisas well as endoscopic analysis can be used to identify intestinalmucositis conditions such as intestinal polyposis. In addition, symptomssuch as diarrhea, abdominal cramps, gas, and nausea can indicateinflammation of the intestines.

In general, a non-invasive fungus-induced intestinal mucositis conditioncan be identified by the presence of fungal organisms, eosinophils, oreosinophil products within stool samples. In addition, tissue biopsiesrevealing the presence of non-invasive fungal organisms can indicate anon-invasive fungus-induced intestinal mucositis condition.

Any individual that had a previous episode of an intestinal inflammatorycondition is at risk for developing non-invasive fungus-inducedintestinal mucositis. In addition, elderly individuals as well asindividuals having a family history of digestive problems, intestinalpolyposis, or allergies can be at risk for developing non-invasivefungus-induced intestinal mucositis.

Fungal Organisms

Any fungal organism living in the mucus of a mammal can be anon-invasive fungal organism that is capable of inducing mucositis sinceit is the mere presence of the organism in an intolerant individual'smucus that causes inflammation. For example, all fungal organismspreviously identified in mucus samples of AFS patients can benon-invasive fungal organisms capable of inducing non-invasivefungus-induced mucositis including, without limitation, Absidia,Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus,Aspergillus nidulans, Aspergillus versicolor, Alternaria, Basidiobolus,Bipolaris, Candida albicans, Candida lypolytica, Candida parapsilosis,Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschlera,Exserohilum, Fusarium, Malbranchia, Paecilomvces, Penicillium,Pseudallescheria, Rhizopus, Schizophylum, and Sporothrix. In addition,fungal organisms that were, until now, not identified in mucus samplesof patients diagnosed positive for AFS can be non-invasive fungalorganisms capable of causing a non-invasive fungus-induced mucositisincluding, without limitation, Acremonium, Arachniotus citrinus,Aurobasidioum, Beauveria, Chaetomium, Chryosporium, Epicoccum, Exophiliajeanselmei, Geotrichum, Oidiodendron, Phoma, Pithomyces, Rhinocladiella,Rhodoturula, Sagrahamala, Scolebasidium, Scopulariopsis, Ustilago,Trichoderma, and Zygomycete. A list of additional fungal organisms thatcan be non-invasive fungal organisms capable of inducing a non-invasivefungus-induced mucositis can be found in most taxonomic mycology textbooks.

Collecting Mucus Samples

In general, mucus can be collected from the surface of any mucosaltissue by using a collection solution to flush the mucus-containingcavity. Proper mucus collection techniques should maximize recovery of amucus-containing collection solution by allowing sufficient penetrationof the appropriate anatomic cavities and by minimizing collectionsolution absorption by the individual. Vasoconstrictor agents can beused to maximize mucus collection and mucolytic agents can be used todissolve obstructive mucus such that collection solution penetration isenhanced.

Thus, before collecting a mucus sample, an individual can be treatedwith a vasoconstrictor agent and/or a mucolytic agent such thatsufficient vasoconstriction and/or mucolytic action is induced in theappropriate region. Suitable vasoconstrictor agents can include, withoutlimitation, phenylephrine hydrochloride (NEO-SYNEPHRINE®; SanofiPharmaceuticals), cocaine, and epinephrine. A mucolytic agent is anyagent that liquefies mucus such that it can be recovered from thepatient. Suitable mucolytic agents can include, without limitation,N-acetyl-L-cysteine (MUCOSIL™; Dey Laboratories) and recombinant humanDNase (PULMOZYME®, Genentech, Inc.). Any administered vasoconstrictoragent or mucolytic agent should be allowed to take effect by waiting asufficient period of time after administration such as about two to fiveminutes.

The following methods and materials can be used to collect anasal-paranasal mucus sample. First, an individual is prepared toreceive a collection solution in at least one nostril or nasal-paranasalcavity by directing the individual to inhale and to lower the chin, orin some other way constrict the access of fluids out of the mouth anddown the esophagus. In a vertically sitting or standing individual,these maneuvers tend to minimize the loss or ingestion of the collectionsolution. Other maneuvers are also possible provided this goal isachieved. Second, an injection and collection system is configured. Ingeneral, the configuration is such that a collection solution can beadministered to an individual's nostril and then efficiently collectedin a container. The injection system can be, without limitation, asyringe with a curved blunt needle or tube assembly. The container canbe any type of container that holds liquid. In addition, the containercan be, without limitation, a storage container that is suitable for useas a transporter or sealable apparatus such that the collected samplecan be handled or shipped. These containers also can contain an agentsuch as a preservative or antibacterial agent depending upon the desireduse of the mucus sample. Third, a collection solution is administeredinto an individual's nostril and collected. Before administration, theindividual can be instructed to expel the collection solution uponsensing the fluid in its nasal-paranasal anatomy. Alternatively, theindividual can be instructed to expel the collection solutionsimultaneously with the administration. During administration, thecollection solution can be forcibly injected into at least one nostrilor side of the nasal-paranasal anatomy. The volume of the collectionsolution can vary according to the individual and the state of themucositis. For example, fluid volumes can be, without limitation,between about 0.1 mL to about 100 mL or more, and specifically betweenabout 0.1 mL and about 25 mL. The collection solution can be, withoutlimitation, a saline solution, water, and any other suitable solutionappropriate for contacting mucosal tissue. In addition, the collectionsolution can contain other agents that may be useful for the collectionof mucus such as a mucolytic agent.

One goal of a collection solution is to dislodge and remove mucus withinmucus-containing cavities. In addition to a collection solution actingas a natural flushing agent, the penetrating effect of a mucolytic agentwithin a collection solution can help liquefy thick obstructive mucus.Further, the combination of the force of administration with the nearsimultaneous pressurized expulsion by an individual can help dislodgeand collect mucus. Typically, a collection solution can be administeredduring a period of less than about 5 seconds per side. In addition, acollection solution can be administered during a period of less thanabout 3 seconds. Alternatively, the time period of collection solutionadministration can be extend beyond 5 seconds depending on specificfactors such as the degree of inflammation, the presence ofobstructions, and the size of the individual. In addition, a greaterthan 5 second administration can be used when very small volumes orstreams of collection solution are desired.

Other collection procedures also can be used to collect mucus samples,particularly if an individual is unable to comply or cope with a liquidcollection procedure. Such additional procedures are well known in theart and include, without limitation, the surgical removal of mucus, aswab or mechanical mucus extraction procedure, and pressure or vacuumsystems that extract mucus. In addition, these other collectionprocedures as well as the methods and materials described herein can bemodified or adapted to obtain biological fluids from other areas of thebody such as the middle ear and intestines.

After a mucus sample is collected, the sample can be analyzed for thepresence of markers that indicate the involvement of non-invasivefungus-induced mucositis. For example, a mucus sample can be examined todetermine the presence of allergic mucus. In addition, fungal organismscan be cultured and analyzed from a mucus sample using the techniquesdescribed herein as well as those techniques known in the art.

FIGS. 3 and 4 depict an exemplary device 10 for aspirating andcollecting mucus and other liquids. Device 10 includes upper member 12,collection retainer 14, and collection tube 16. Upper member 12, inturn, broadly includes central portion 22, threaded portion 24,connecting portion 26, and tube receiving member 28. Central portion 22may generally define opening 29 therein. Valve 30 is operably disposedwithin opening 29. Threaded portion 24 may extend downwardly fromcentral portion 22. Connecting portion 26 extends radially from centralportion 22, is generally circular in cross-section in this embodiment,and defines bore 32. Bore 32 communicates the exterior of device 10 withan interior portion thereof. Tube receiving member 28 extends generallyradially from central portion 22. Tube receiving member 28 is disposedgenerally opposite connection portion 26 in this embodiment. Tubereceiving member 28 defines bore 34. As with bore 32, bore 34communicates the exterior of device 10 to the interior thereof.

In this embodiment, retainer 14 is threadably received onto threadedportion 24. Retainer 14, however, may be secured to central portion 22by other known means. Although a conically-shaped bottom configurationis depicted, retainer 14 may assume a variety of configurations and bewithin the scope of this invention. Collection tube 16 extends from andis disposed within bore 34. A length of tube 16 may be disposed withinthe interior of retainer 14 to facilitate placement of collectedmaterial. Collection tube 16 defines lumen 36 through which thecollected mucus travels. In one embodiment, tube 16 comprises a flexiblememory means to facilitate adaptation to different patient anatomies.That is, tube 16 remains conformed to a desired and flexibleconfiguration, for example, as depicted by the phantom lines in the FIG.3. Further means to facilitate travel of the mucus through thecollection tube and device 10 may include a tube or device liner havingmaterial characteristics designed to minimize adherence of the mucusthereto.

In one embodiment, device 10 is designed for a single use. Device 10 maybe made from a number of materials, however, synthetic resins such aspolyethylene may be used. Connecting portion 26 connects device 10 to avacuum hose 38. Thus, connecting portion 26 may have an outerconfiguration such that an air tight fit to vacuum hose 38 results.Valve 30 adjusts the amount of vacuum communicated through lumen 36. Byadjusting valve 30, a gradually increasing or decreasing amount ofvacuum may be applied thereto. In this example, valve 30 includes agenerally elongate slit which is configured with a sliding member. Thesliding member may be adjusted by the user so that all, none, or aportion of the elongate slit is exposed, thereby adjusting the vacuumcommunicated to lumen 36. A variety of other adjustment means forregulating the vacuum, however, are within the scope of this invention.Another example includes an “IV” type of valve which utilizes a rollervalve to adjustably constrict a draw or collection tube.

In contrast to other devices, tube receiving member 28 and collectiontube 16 extend generally perpendicularly from a longitudinal axis ofretainer 14. This enables the users to better position collection tube16 when recovering mucus and other liquids. It is recognized that othercollection containers are possible within the scope of this inventionwhich conform comfortably to a patient's facial anatomy, and which maybe readily held in place by either the patient or a health careprovider. Such embodiments may rely on vacuum, gravity, or othercollection mechanisms provided they afford ready access for fluids beinginjected into the patient while simultaneously allowing drainage orwithdrawal of the fluids and mucus from the patient.

In this embodiment lumen 36 is between about 1 mm and 10 mm in diameter,and between about 5 cm and 50 cm in total length. Exemplary retainer 14is generally between about 1″ to 3″ in diameter and 3″ to 6″ in height,although various other sizes may be useful.

Device 10 is advantageously utilized to obtain mucus or fluid samplesfrom a patient's nasal, paranasal, or pulmonary anatomy. In obtainingmucus or fluid samples, device 10 is connected to a vacuum source andvalve 30 is adjusted as desired. Tube 16 is configured to a desiredposition. Tube 16 is then inserted into a portion of the patient'sanatomy from which mucus or fluid is to be obtained. Valve 30 is furtheradjusted as necessary to obtain the sample, yet assure safety to thepatient. The obtained mucus or fluid is collected in retainer 14. Oncecollection is complete, retainer 14 may be detached from upper member 12for storage or shipment of the obtained mucus or fluid sample.

Culturing Fungal Organisms from a Mucus Sample

A mucus sample can be prepared for fungal organism culturing by treatingthe sample with a mucolytic agent such as N-acetyl-L-cysteine ordithiothreitol (DTT) to enhance or facilitate further liquefaction ofmucus. After adding a mucolytic agent, the mucus sample can be mixed andincubated at room temperature. This liquefaction allows fungal organismspresent within mucus to be released. Once liquefied, mucus can beisolated by centrifugation or other means since mucus typically forms alayer separate from the other solutions (i.e., collection solution).Once isolated, the mucus can be mixed and an aliquot placed in contactwith an appropriate fungal growth medium such as growth medium agarplates. A fungal growth medium is any medium that can support the growthof a fungal organism including, without limitation, RPMI-1649,Delbecco's modified eagles medium (DMEM), inhibitory mold agar (IMA),and Bay agar. The fungal growth medium can contain antibacterial agents(e.g., chloramphenicol and ciprofloxacin) to prevent the growth ofbacteria.

Once liquefied mucus is placed in contact with an appropriate fungalgrowth medium, the cultures can be incubated at an optimal temperature,for example, between about 20° C. and 37° C. and, in some instances,between about 25° C. and 35° C. An optimum temperature can be assessedby placing duplicate cultures at various temperatures and comparinggrowth rates. Typically, cultures are incubated between about two tothirty-five days at about 30° C. Once fungal growth is observed, thefungal species can be identified using procedures well known in the artand the phenotype and genotype of each fungal isolate characterized. Forexample, a fungal isolate can be examined to determine any drugresistant or drug susceptibility properties.

Treating and Preventing Non-invasive Fungus-induced Mucositis

Antifungal agents can be mucoadministered to a mammal in an amount, at afrequency, and for a duration effective to treat or prevent non-invasivefungus-induced mucositis. An “antifungal agent” is any agent that isactive against a fungal organism. For example, an antifungal agent isany agent that prevents the growth of or kills a fungal organism such asantifungal polyene macrolides, tetraene macrolides, pentaenicmacrolides, fluorinated pyrimidines, imidazoles, triazoles, azoles,halogenated phenolic ethers, thiocarbamates, and allylamines. Inaddition, antifungal agents can be agents that interpolate fungal cellwall components or act as sterol inhibitors. Specific antifungal agentswithin the scope of the invention include, without limitation,amphotericin B, flucytosine, ketoconazole, miconazole, itraconazole,fluconazole, griseofulvin, clotrimazole, econazole, terconazole,butoconazole, oxiconazole, sulconazole, saperconazole, voriconazole,ciclopirox olamine, haloprogin, tolnaftate, naftifine, nystatin,natamycin, terbinafine hydrochloride, morpholines, butenafineundecylenic acid, Whitefield's ointment, propionic acid, and caprylicacid as well as those agents that can be identified as antifungal agentsusing methods well known in the art. It is noted that a particularpatient may possess a fungal organism acting as the etiological agentthat is resistant to a particular antifungal agent. In such a case, animportant aspect of this invention involves treating that patient withan effective antifungal agent (e.g., an antifungal agent that preventsthe growth of, or kills, the fungal organism acting as the etiologicalagent). Such fungal organisms acting as etiological agents can beidentified using the collection and culture methods described herein.

The term “mucoadministration” as used herein refers to any type ofadministration that places an administered agent in contact with mucus.Thus, any intravenously administered agent that does not exit the bloodstream is not considered a mucoadministered agent because the agentfailed to contact mucus. In addition, the term “mucoadministration” canbe subdivided into “direct” and “indirect” mucoadministration. The term“direct mucoadministration” as used herein refers to any type ofadministration that places an administered agent in direct contact witha targeted mucus prior to crossing epithelium. For the purpose of thisinvention, it is to be understood that injections of an agent into acavity containing mucus is considered direct mucoadministration if theagent contacts mucus even though an injection means (e.g., needle, tube,or catheter) may be used to cross an epithelium. Thus, using a needle tobypass the tympanic membrane and inject an agent into the middle ear isconsidered a direct mucoadministration that targets middle ear mucus.

It follows that any intravenously administered agent that subsequentlyexits the blood stream, permeates epithelium, and contacts mucus is notconsidered a directly mucoadministered agent because the agent crossedepithelium prior to contacting mucus. In this case, however, theintravenously administered agent is considered an indirectlymucoadministered agent since the term “indirect mucoadministration”means any type of administration that places an administered agent incontact with a targeted mucus after crossing epithelium. Again, the useof an injection means such as a needle, tube, or catheter to deliver anagent past epithelium and into direct contact with mucus does notnecessarily mean the administration is an indirect mucoadministration.

It also follows that an oral administration can be either a direct orindirect mucoadministration depending on the targeted mucus. Forexample, an agent can be swallowed and then traverse the esophagus,stomach, and small intestine to come in direct contact with mucus in thelarge intestine, without having crossed an epithelium (i.e., directmucoadministration). At the same time, the orally administered agentcould be absorbed by the gut, accumulate systemically, and permeate thenasal epithelium to come in contact with nasal mucus (i.e., indirectmucoadministration). Thus, the direct and indirect nature ofmucoadministration depends upon the specific route of administration aswell as the specific location of the targeted mucus. Typical routes ofdirect and indirect mucoadministration for various mucus locations of amammal are described below.

An effective amount of an antifungal agent or formulation containing anantifungal agent can be any amount that reduces, prevents, or eliminatesnon-invasive fungus-induced mucositis upon mucoadministration in amammal without producing significant toxicity to the mammal. Typically,an effective amount can be any amount greater than or equal to theminimum inhibitory concentration (MIC) for a fungal organism or isolatepresent within a particular individual's mucus that does not inducesignificant toxicity to the individual upon mucoadministration. Someantifungal agents may have a relatively large concentration range thatis effective while others may have a relatively narrow effectiveconcentration range. In addition, the effective amount can varydepending upon the specific fungal organism or isolate since certainorganisms and isolates are more or less susceptible to particularantifungal agents. Such effective amounts can be determined forindividual antifungal agents using commonly available or easilyascertainable information involving antifungal effectivenessconcentrations, animal toxicity concentrations, and tissue permeabilityrates. For example, non-toxic antifungal agents typically can bedirectly or indirectly mucoadministered in any amount that exhibitsantifungal activity within mucus. In addition, antifungal agents that donot permeate mucosal epithelium typically can be directlymucoadministered in any amount that exhibits antifungal activity withinmucus. Using the information provided herein, such effective amountsalso can be determined by routine experimentation in vitro or in vivo.For example, a patient having a non-invasive fungus-induced mucositiscondition can receive direct mucoadministration of an antifungal agentin an amount close to the MIC calculated from in vitro analysis. If thepatient fails to respond, then the amount can be increased by, forexample, ten fold. After receiving this higher concentration, thepatient can be monitored for both responsiveness to the treatment andtoxicity symptoms, and adjustments made accordingly.

For amphotericin B, an effective amount can be about 0.01 ng to about1000 mg per kg of body weight of the mammal per administration whendirectly mucoadministered. When used as a nasal irrigation solution, aneffective amount can be a volume of about 0.01 mL to about 1 liter pernostril per administration of a solution containing about 0.01 mg ofamphotericin B per liter to about 1000 mg of amphotericin B per liter.Alternatively, an effective amount can be 20 mL per nostril peradministration (e.g., two to four times daily) of an irrigation solutioncontaining about 100 mg of amphotericin B per liter of saline or water.Typically, the saline or water is sterile. The effective amount canremain constant or can be adjusted as a sliding scale or variable dosedepending on the individual's response to treatment. Effective amountsfor other antifungal agents can be determined by a person of ordinaryskill in the art using routine experimentation in view of the multipleteachings described herein.

Typically, an effective amount of any antifungal agent directlymucoadministered (e.g., itraconazole, ketoconazole, and voriconazole)can be about 0.01 ng to about 1000 mg per kg of body weight of themammal per administration. The MIC values for voriconazole range fromabout 0.003 μg/mL to about 4 μg/mL depending upon the specific fungalorganism or isolate tested. For fluconazole, the MIC values range fromabout 0.25 μg/mL to greater than about 64 μg/mL.

To help determine effective amounts of different antifungal agents, itcan be useful to refer to an effective amount equivalent based on theeffective amount of a common antifungal agent. For example, the directmucoadministration of about 20 mL per nostril per administration (e.g.,twice daily) of an amphotericin B irrigation solution containing about100 mg of amphotericin B per liter is an effective amount asdemonstrated herein. The effects produced by this effective amount canbe used as a reference point to compare the effects observed for otherantifungal agents used at varying concentrations. Once an equivalenteffect is observed, then the specific effective amount for thatparticular antifungal agent can be determined. In this case, thatparticular amount would be termed an amphotericin B effective amountequivalent.

Various factors can influence the actual effective amount used for aparticular application. For example, the frequency ofmucoadministration, duration of treatment, combination of otherantifungal agents, site of administration, degree of inflammation, andthe anatomical configuration of the treated area may require an increaseor decrease in the actual effective amount mucoadministered.

The frequency of mucoadministration can be any frequency that reduces,prevents, or eliminates non-invasive fungus-induced mucositis in amammal without producing significant toxicity to the mammal. Forexample, the frequency of mucoadministration can be from about fourtimes a day to about once a month, or more specifically, from abouttwice a day to about once a week. In addition, the frequency ofmucoadministration can remain constant or can be variable during theduration of treatment. As with the effective amount, various factors caninfluence the actual frequency of mucoadministration used for aparticular application. For example, the effective amount, duration oftreatment, combination of other antifungal agents, site ofadministration, degree of inflammation, and the anatomical configurationof the treated area may require an increase or decrease inmucoadministration frequency.

An effective duration for antifungal agent mucoadministration can be anyduration that reduces, prevents, or eliminates non-invasivefungus-induced mucositis in a mammal without producing significanttoxicity to the mammal. Thus, the effective duration can vary fromseveral days to several weeks, months, or years. In general, theeffective duration for the treatment of non-invasive fungus-inducedmucositis can range in duration from several days to several months.Once the antifungal applications are stopped, however, non-invasivefungus-induced mucositis may return. Thus, the effective duration forthe prevention of non-invasive fungus-induced mucositis can last in somecases for as long as the individual is alive.

For anatomies that are less susceptible to fungal organism repopulationfactors (e.g., a human middle ear with an intact tympanic membrane), aneffective duration can range from about 10 days to about 30 days. Forless sterile environments such as the nasal-paranasal anatomy, however,an effective duration can range from about 30 days to greater than about80 days. In the respiratory tract or digestive tract, an effectiveduration can be from about 10 days to greater than about 30 days, oreven greater than about 90 days. Again, prophylactic treatments aretypically longer in duration and can last throughout an individual'slifetime.

Multiple factors can influence the actual effective duration used for aparticular treatment or prevention regimen. For example, an effectiveduration can vary with the frequency of antifungal agent administration,effective antifungal agent amount, combination of multiple antifungalagents, site of administration, degree of inflammation, and anatomicalconfiguration of the treated area. Further, the specific antifungalagent used can influence the actual effective duration. For example, aneffective duration for treating non-invasive fungus-inducedrhinosinusitis can be about 30 days for amphotericin B and about 7 daysfor itraconazole.

It is noted that diagnostic algorithm methods can be devised todetermine or reflect appropriate effective doses, durations, andfrequencies.

Formulations Containing at Least One Antifungal Agent

A formulation containing an antifungal agent can be in any form providedthe formulation can be mucoadministered to a mammal in an amount, at afrequency, and for a duration effective to prevent, reduce, or eliminatea non-invasive fungus-induced mucositis. For example, a formulationwithin the scope of the invention can be in the form of a solid, liquid,and/or aerosol including, without limitation, powders, crystallinesubstances, gels, pastes, ointments, salves, creams, solutions,suspensions, partial liquids, sprays, nebulae, mists, atomized vapors,tinctures, pills, capsules, tablets, and gelcaps. In addition, theformulation can contain a cocktail of antifungal agents. For example, aformulation within the scope of the invention can contain, withoutlimitation, one, two, three, four, five, or more different antifungalagents. Further, formulations within the scope of the invention cancontain additional ingredients including, without limitation,pharmaceutically acceptable aqueous vehicles, pharmaceuticallyacceptable solid vehicles, steroids, mucolytic agents, antibacterialagents, anti-inflammatory agents, immunosuppressants, dilators,vaso-constrictors, decongestants, leukotriene inhibitors,anti-cholinergics, anti-histamines, therapeutic compounds, andcombinations thereof. In addition, a formulation can contain any one ormore compounds known to be effective for inhibiting the gag reflex of amammal.

A pharmaceutically acceptable aqueous vehicle can be, for example, anyliquid solution that is capable of dissolving an antifungal agent and isnot toxic to the particular individual receiving the formulation.Examples of pharmaceutically acceptable aqueous vehicles include,without limitation, saline, water, and acetic acid. Typically,pharmaceutically acceptable aqueous vehicles are sterile. Apharmaceutically acceptable solid vehicle can be formulated such thatthe antifungal agent is suitable for oral administration. For example,capsules or tablets can contain an antifungal agent in enteric form. Thedose supplied by each capsule or tablet can vary since an effectiveamount can be reached by administrating either one or multiple capsulesor tablets. Any well known pharmaceutically acceptable material such asgelatin and cellulose derivatives can be used as a pharmaceuticallyacceptable solid vehicle. In addition, a pharmaceutically acceptablesolid vehicle can be a solid carrier including, without limitation,starch, sugar, or bentonite. Further, a tablet or pill formulation of anantifungal agent can follow conventional procedures that employ solidcarriers, lubricants, and the like.

Steroids can be any compound containing a hydrocyclopentanophenanthrenering structure. Examples of steroids include, without limitation,prednisone, dexamethasone, and hydrocortisone. Mucolytic agents can beany compound that liquefies mucus. Suitable mucolytic agents caninclude, without limitation, N-acetyl-L-cysteine (MUCOSIL™; DeyLaboratories) and recombinant human DNase (PULMOZYME®; Genentech, Inc.).An antibacterial agent can be any compound that is active againstbacteria, such as penicillin, erythromycin, neomycin, gentamicin, andclindamycin. An anti-inflammatory agent can be any compound thatcounteracts inflammation, such as ibuprofen and salicylic acid. Animmunosuppressant can be any compound that suppresses or interferes withnormal immune function, such as cyclosporine. A dilator can be anycompound that causes the expansion of an orifice, such as albuterol. Avaso-constrictor can be any compound that constricts or narrows bloodvessels, such as phenylephrine hydrochloride (NEO-SYNEPHRINE®; SanofiPharmaceuticals), cocaine, and epinephrine. A decongestant can be anycompound that acts to reduce nasal-paranasal congestion or swelling,such as pseudoephedrine hydrochloride, phenylpropanolamine, andoxymetazoline. A leukotriene inhibitor can be any compound that inhibitsthe function or synthesis of a leukotriene, such as Azelastine®. Ananti-cholinergic can be any compound that blocks parasympathetic nerveimpulses, such as ipratropium bromide. An anti-histamine can be anycompound that opposes the action of histamine or its release from cells(e.g., mast cells), such as terfenadine and astemizole.

A therapeutic compound can be any compound that has a therapeutic effectupon administration. For example, a therapeutic compound can be anycompound that blocks or interferes with the interaction of an eosinophilwith an immunoglobulin bound to a fungal antigen by targeting, forexample, Fc receptor or S-type lectin factor receptor (e.g., galectin-3)interactions. Such compounds can include, without limitation, antibodiessuch as IgE, IgA, IgG, IgM, and IgD as well as antibody fragments suchas Fab, F(ab′)₂, FcγRI, FcγRII, FcαR, FcεRII, and FcεRI.

Mucoadministration Targeting the Nasal-paranasal Anatomies

The mucoadministration of an agent to the nasal-paranasal anatomies canbe any type of administration that places the agent in contact withnasal-paranasal mucus. Direct mucoadministration to the nasal-paranasalanatomies can include, without limitation, nasal irrigations, nasalsprays, nasal inhalations, and nasal packs with, for example, saturatedgauze provided the administered agent contacts nasal-paranasal mucusprior to crossing epithelium. In addition, injections into thenasal-paranasal cavities using, for example, a needle or catheter tubeis considered a direct mucoadministration provided the administeredagent contacts nasal-paranasal mucus after leaving the needle orcatheter tube and prior to crossing epithelium. Any device can be usedto directly mucoadminister an agent to the nasal-paranasal anatomyincluding, without limitation, a syringe, bulb, inhaler, canister, spraycan, nebulizer, and mask. For example, a 20 mL bulb can be used toirrigate the nasal-paranasal anatomy with a liquid form of a formulationcontaining an antifungal agent. Such a liquid form of a formulation canbe stored at −20° C., 0° C., or room temperature. If stored below roomtemperature, the formulation typically is warmed prior to application tothe nasal/paranasal cavities.

Indirect mucoadministration to the nasal-paranasal anatomies caninclude, without limitation, oral, intravenous, intradermal, andintraperitoneal administrations provided the administered agent contactsnasal-paranasal mucus. In addition, any device can be used to indirectlymucoadminister an agent to the nasal-paranasal anatomy including,without limitation, a syringe and regulated release capsule.

It is noted that the particular route of administration can influencethe effective amount and duration of treatment with antifungal agents aswell as the frequency of mucoadministration. For example, orallymucoadministered antifungal agents may require higher concentrations todeliver an effective amount to nasal-paranasal mucus than directmucoadministration by nasal irrigations.

Mucoadministration Targeting the Lung Airways

An airway is any part of the mammalian anatomy that air traverses duringrespiration including the mouth, nasal passages, trachea, bronchi, andbronchial tubes. A lung airway is any air passage of the lung lined bymucosa including the bronchi and bronchial tubes. The mucoadministrationof an agent to the lung airways can be any type of administration thatplaces the agent in contact with lung airway mucus. Directmucoadministration to the lung airways can include, without limitation,inhalations, nasal sprays, and nasal irrigations provided theadministered agent contacts lung airway mucus prior to crossingepithelium. In addition, injections into lung airways using, forexample, a needle or catheter tube is considered a directmucoadministration provided the administered agent contacts lung airwaymucus after leaving the needle or catheter tube and prior to crossingepithelium. Any device can be used to directly mucoadminister an agentto the lung airway including, without limitation, a syringe, bulb,inhaler, nebulizer, aerosol canister, spray can, and mask.

Indirect mucoadministration to the lung airways can include, withoutlimitation, oral, intravenous, intradermal, and intraperitonealadministrations provided the administered agent contacts lung airwaymucus after crossing epithelium. In addition, any device can be used toindirectly mucoadminister an agent to lung airways including, withoutlimitation, a syringe and regulated release capsule.

It is noted that the particular route of administration can influencethe effective amount and duration of treatment with antifungal agents aswell as the frequency of mucoadministration. For example, entericmucoadministration of an antifungal agent may require a higherconcentration to deliver an effective amount to lung airway mucus thandirect mucoadministration by inhalation through the mouth or nose.

It is to be understood that the direct and indirect mucoadministrationto the airways including the trachea, nasal passages, and mouth can beaccomplished using the methods and material described herein for thelung airways. Mucoadministration Targeting the Middle Ear

The mucoadministration of an agent to the middle ear can be any type ofadministration that places the agent in contact with middle ear mucus.The direct mucoadministration to the middle ear can include, withoutlimitation, ear drops and ear flushes provided the administered agentcontacts middle ear mucus prior to crossing epithelium. For example, ifthe tympanic membrane is damaged or otherwise punctured, then an earflush would be considered a direct mucoadministration provided theadministered agent contacts middle ear mucus. In addition, injectionsinto the middle ear using, for example, a needle or myringotomy tube isconsidered a direct mucoadministration provided the administered agentcontacts middle ear mucus after leaving the needle or tube and prior tocrossing epithelium. Any device can be used to directly mucoadministeran agent to the middle ear including, without limitation, a syringe andbulb.

Indirect mucoadministration to the middle ear can include, withoutlimitation, oral, intravenous, intradermal, and intraperitonealadministrations provided the administered agent contacts middle earmucus after crossing epithelium. In addition, any device can be used toindirectly mucoadminister an agent to the middle ear including, withoutlimitation, a syringe and regulated release capsule.

It is noted that the particular route of administration can influencethe effective amount and duration of treatment with antifungal agents aswell as the frequency of mucoadministration. For example, orallymucoadministered antifungal agents may require higher concentrations todeliver an effective amount to middle ear mucus than directmucoadministration by middle ear injection.

Mucoadministration Targeting the Intestines

The mucoadministration of an agent to the intestines can be any type ofadministration that places the agent in contact with intestinal mucus.The direct mucoadministration to the intestines can include, withoutlimitation, oral and enema administrations provided the administeredagent contacts intestinal mucus prior to crossing epithelium. Inaddition, injections into the digestive tract using, for example, aneedle or catheter tube is considered a direct mucoadministrationprovided the administered agent contacts intestinal mucus after leavingthe needle or catheter tube and prior to crossing epithelium. Any devicecan be used to directly mucoadminister an agent to the intestinesincluding, without limitation, a syringe and regulated release capsule.For example, an antifungal agent can be formulated into a regulatedrelease capsule such that the antifungal agent is released afterpassing, for example, the stomach (e.g., pH regulated release capsulesand time regulated release capsules).

Indirect mucoadministration to the intestines can include, withoutlimitation, intravenous, intradermal, and intraperitonealadministrations provided the administered agent contacts intestinalmucus. In addition, any device can be used to indirectly mucoadministeran agent to the intestines including, without limitation, a syringe.

It is noted that the particular route of administration can influencethe effective amount and duration of treatment with antifungal agents aswell as the frequency of mucoadministration. For example, intravenouslymucoadministered antifungal agents may require higher concentrations todeliver an effective amount to intestinal mucus than directmucoadministration by an enema.

Additional Treatments

Other treatments can be used in combination with a formulationcontaining an antifungal agent to help enhance the treatment orprevention of non-invasive fungus-induced mucositis conditions. Suchadditional treatments can include, without limitation, surgeries and theadministration of a second formulation. Surgeries can include, withoutlimitation, the removal of polypoid growths or other tumors, thephysical opening of a cavity, and the insertion of catheter tubes andthe like. A second formulation can include, without limitation,antifungal agents, mucolytic agents, antibacterial agents,anti-inflammatory agents, immunosuppressants, dilators,vaso-constrictors, decongestants, steroids, anti-cholinergics,leukotriene inhibitors, anti-histamines, therapeutic compounds, andcombinations thereof. In addition, this second formulation can beadministered to a mammal by any route. For example, oral,intraperitoneal, intradermal, intravenous, subcutaneous, intramuscular,topical, intranasal, and intrabronchial administration can be used todeliver a second formulation to a mammal.

Treating and Preventing Asthma

Asthma can be characterized by a paradoxical narrowing of the bronchi(lung passageways) such that breathing becomes difficult. Individualssuffering from asthma can exhibit symptoms such as wheezing, difficultybreathing (particularly exhaling air), dyspnea, and tightness in thechest. Factors that can exacerbate asthma include rapid changes intemperature or humidity, allergies, upper respiratory infections,exercise, stress, and smoking. Individuals suffering from asthma can beidentified using any of the known methods in the art. In general, asthmacan be, without limitation, diagnosed objectively with a pulmonaryfunction test (increased airway resistance) with or without provokingthe airway (e.g., methacholine challenge test), chest X-rays, andauscultation of the chest.

Individuals at risk for developing asthma can include, withoutlimitation, those individuals that have had a previous episode ofasthma. In addition, elderly individuals; individuals having cysticfibrosis, chronic rhinosinusitis with or without gross nasal-paranasalpolyps, aspirin sensitivity, or a family history of respiratory problemsor allergies; and individuals that are exposed to significant levels ofallergens (e.g., fungus spores, pollen, and chemicals) or irritants canbe at risk for developing asthma.

Chronic asthmatic individuals can be treated by directlymucoadministering an antifungal agent to at least a portion of theairways in an amount, at a frequency, and for a duration effective toreduce or eliminate asthma symptoms. Such direct mucoadministrations canbe similar to the methods and materials described herein for thetreatment and prevention of non-invasive fungus-induced rhinosinusitissince the nasal-paranasal cavities are airways. For example, nasalsprays and nasal irrigations can be used to directly mucoadministerantifungal agents to airway mucus. In addition, chronic asthmaticindividuals can be treated by directly mucoadministering an antifungalagent to at least a portion of the lung airways in an amount, at afrequency, and for a duration effective to reduce or eliminate asthmasymptoms. For example, aerosol or powder forms of an antifungal agentcan be used for direct mucoadministration to lung airway mucus byinhalation through the mouth or nose.

Further, individuals at risk for developing chronic asthma can beprophylactically treated by mucoadministering an antifungal agent to atleast a portion of the airways in an amount, at a frequency, and for aduration effective to prevent asthma symptoms. Again, such prophylactictreatments can be similar to the methods and materials described hereinfor the prophylactic treatment of non-invasive fungus-inducedrhinosinusitis.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Collecting and Analyzing Mucus Samples

The following methods and materials were used to collect and analyzemucus from 202 patients. Prior to collecting the mucus, each patient wasdirected to inhale and then lower his or her chin toward their chest tominimize or prevent the flow of a collection solution out of thenasal-paranasal passageways via the normal drainage at the back of thethroat. The collection solution was either a sterile saline solution orsterile water. In addition, each patient was positioned such that theflow of the collection fluid out of the nasal passageways would beminimized or prevented. Some patients received an administration of avasoconstrictor, such as phenylephrine hydrochloride (1-2 sprays pernostril) or cocaine (topical liquid or powder; less than four mg per kgof body weight). Some patients received a spray of about three mL of a20% solution of N-acetyl-L-cysteine. Patients receiving both were giventhe vasoconstrictor first and then about two minutes later givenN-acetyl-L-cysteine.

Once the patient was prepared, a collection container was placed underthe nostril or nostrils from which the mucus sample was to be collected.An injection device, such as a syringe-like device having a tubeassembly or blunt curved needle, was then partially placed into one ofthe patient's nostrils or paranasal anatomy such that the collectionsolution could be forced through the patient's nasal-paranasal anatomy.In some cases, about five mL to about 30 mL of a collection solution wasthen injected into a nostril during a time period of about 0.5 and fiveseconds. In most cases, about ten mL to about 20 mL of a collectionsolution was injected into a nostril during a time period of betweenabout 0.5 and three seconds.

In general, each patient blew out or forcefully discharged thecollection solution either simultaneously as it was being injected orupon sensing its entry into the nostril. This forceful discharging ofthe injected collection solution contributed significantly to looseningmucus within the patient's nasal and paranasal lumens. Again, specialcare was taken to reduce or prevent the loss of collection solutionvolume. Once expelled, the collection solution containing mucus from thepatient's nostril was collected in the collection container placed underthe nostril. After nasal-paranasal mucus was collected, the mucus wascultured using one of the following two methods. In the first method,one mL of a 20% solution of N-acetyl-L-cysteine was added to about tenmL of the recovered collection solution containing mucus. This mixturewas then vortexed for 30 seconds and incubated for 15 minutes at roomtemperature. After incubation, the mixture was centrifuged in a 50 mLtube for five minutes at 4800 rpm. After separation, the supernatant wasdiscarded and the remaining mucus vortexed for 30 seconds. A 0.5 mLaliquot of the isolated mucus was then added to each culture plate, oneIMA plate containing chloramphenicol and one IMA plate containingciprofloxacin. The plates were then incubated at 30° C. and read as aroutine fungal culture. Growth of individual isolates was observed fromabout two days to about 35 days.

In the second method, ten mL of DTT was diluted with 90 mL of steriledistilled water. An equal volume of this freshly diluted DTT solutionwas added to the recovered collection solution containing mucus and themixture vortexed for 30 seconds. This mixture was then incubated for 15minutes at room temperature. After incubation, the mixture wascentrifuged in a 50 mL tube for ten minutes at 3000×g. After separation,the supernatant was discarded and the remaining mucus vortexed for 30seconds. A 0.5 mL aliquot of the isolated mucus was then added to eachculture plate, one IMA plate containing chloramphenicol and one Bay agarplate containing ciprofloxacin. The plates were then incubated at 30° C.and read as a routine fungal culture. Growth of individual isolates wereobserved from about two days to about 35 days.

Once fungal growth was observed, the organisms were identified usingstandard mycology techniques including visual, histological, andimmunological techniques. Identified fungal genera and species includedmany fungal organisms previously isolated from AFS patients, such asAbsidia, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus,Aspergillus nidulans, Aspergillus versicolor, Alternaria, Basidiobolus,Bipolaris, Candida albicans, Candida lypolytica, Candida parapsilosis,Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschlera,Exserohilum, Fusarium, Malbranchia, Paecilomvces, Penicillium,Pseudallescheria, Rhizopus, Schizophylum, and Sporothrix. In addition,fungal organisms were identified that were not previously identified inmucus samples of patients diagnosed positive for AFS, such asAcremonium, Arachniotus citrinus, Aurobasidioum, Beauveria, Chaetomium,Chryosporium, Epicoccum, Exophilia jeanselmei, Geotrichum, Oidiodendron,Phoma, Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala,Scolebasidium, Scopulariopsis, Ustilago, Trichoderma, and Zygomycete.

To determine the optimum temperature for culturing fungal organisms thatcause non-invasive fungus-induced mucositis, liquefied mucus samplescollected from two patients were cultured onto IMA plates containingeither chloramphenicol or ciprofloxacin. Two dishes (one containingchloramphenicol and one containing ciprofloxacin) for each sample werethen incubated at 25° C., 28° C., 30° C., 32° C., 33° C., 35° C., and37° C. Each plate was visually scored for fungal growth and developmentevery other day over a period from about two days to about 35 days fromthe time of culturing. The scores for each temperature were averaged,thereby providing an estimate of the optimum temperature for sporegermination and subsequent growth or development of fungal organisms.The results indicated that the optimum temperature for fungal growthvaried depending upon the specific fungal species or isolate. Ingeneral, 30° C. was found to support growth for the largest number offungal species and isolates.

The following procedure was used to determine effective antifungalagents as well as effective concentrations of antifungal agents suchthat the fungal organisms isolated from patients can be prevented fromgrowing or killed.

Seventeen fungal isolates were collected from eight rhinosinusitispatients and tested for susceptibility against amphotericin B,ketoconazole, and itraconazole. Each antifungal agent was tested onthese fungal isolates using the macro broth dilution technique accordingto the National Committee on Clinical Laboratory Standards (NCCLS)protocol. A 48 hour MIC reading was recorded and interpreted using NCCLSguidelines to rate each culture as susceptible, intermediate, orresistant to the antifungal agent at the concentrations being tested.The results from this procedure provided an estimate of the efficacy ofantifungal agents against specific fungal isolates in vitro. In general,the MIC values for these antifungal agents for each isolate were foundto range widely between 0.03 and 100 μg/mL (Table I).

TABLE I Fungal organisms isolated from 64 of 66 rhinosinusitis patientsstudied including 17 amphotericin B, ketoconazole, and itraconazole MICvalues for selected isolates from eight rhinosinusitis patients.Amphoter- Keto- Itra- Number of icin B conazole conazole Fungal Species/MIC MIC MIC Organism Isolates (μg/mL) (μg/μL) (μg/mL) Acremonium 1Alternaria 40 0.2 1.56  neg* 0.8 3.13 neg >5 12.5 neg Arachniotus 2citrinus Aspergillus 21 Aurobasidium 2 Candida 13 0.2 50 0.03 0.4 1.56neg 0.1 0.39 neg 0.2 >100 neg Cladosporium 21 3 1.56 neg 0.4 0.05 neg0.1 0.05 0.125 Chryosporium 1 Epicoccum 6 Exophilium 2 JeanselmeiFusarium 18 >5 >100  37° C.** 2 100 1 >5 12.5 >16 Geotrichum 5 0.1 0.0537° C. Mucor 2 Oididendron 1 Paecilomyces 2 lilacinus Papularia 1Penicillium 30 0.4 3.13 0.5 1 1.56 0.25 Phoma 1 Pithomyces 2 Rhodoturula1 Scolebasidium 1 Trichoderma 3 4 50 37° C. Ustilago 2 not identified 7(2 monila, 3 dermatiaceous) *, neg means the organism did not grow inthe test medium (PEG 400); **, 37° C. means the organism did not grow at37° C.

The following study was conducted to determine the frequency ofrhinosinusitis conditions having a non-invasive fungal etiology. Forthis study, the following criteria were used to determine if a patienthad non-invasive fungus-induced rhinosinusitis: (1) presence ofobservable disease within the nasal-paranasal anatomy, (2) presence ofallergic mucus, and (3) presence of fungal organisms withinnasal-paranasal mucus. Each patient had a CT scan using standardprocedures to determine the presence of observable disease within theirnasal-paranasal anatomy. To determine the presence of allergic mucus, asurgical specimen was collected from each patient and evaluatedhistologically. It is noted that special care was used to collect eachsurgical specimen to ensure that mucus samples were not washed away. Todetermine the presence of fungal organisms within nasal-paranasal mucus,the methods and materials for collecting and culturing fungal organismsfrom a patient's mucus described herein were used.

Seventy-three rhinosinusitis patients were entered into the study. Theages of these patients ranged from 13 to 73 years, averaging 50.1 yearsof age. Thirty-nine of the 73 patients were female and 34 were male. Thenumber of previous surgeries relating to rhinosinusitis for each patientranged from 0 to 25, with an average of 3.41 surgeries per patient.Seventy of the 73 patients had previously experienced a recurrence ofpolyposis and rhinosinusitis.

Seven patients were subsequently excluded from the study due to a lackof an acceptable mucus specimen. Of the remaining 66 patients, 66 (100%)were diagnosed as CT-scan-positive, 62 (94%) were diagnosed positive forthe presence of allergic mucus, and 64 (97%) had positive fungalcultures. Taken together, 60 of 66 (91%) cases of rhinosinusitis had allthree criteria. In other words, 91 percent of the 66 rhinosinusitispatients evaluated have, based on the above criteria, non-invasivefungus-induced rhinosinusitis. This 91 percent proportion represents adramatic increase in the number of rhinosinusitis cases involvingnon-invasive fungal organisms. For example, numerous medical researcharticles report that about three to eight percent of chronicrhinosinusitis cases requiring surgery are AFS cases, a rhinosinusitiscondition having a non-invasive fungal etiology. Thus, the resultspresented herein indicate that the involvement of non-invasive fungalorganisms in rhinosinusitis conditions is much more prevalent thanpreviously appreciated.

A total of 25 different fungal species was identified from mucusspecimens from these non-invasive fungus-induced rhinosinusitispatients. Sixteen organisms never before described as present coincidentwith AFS were detected from the 64 mucus samples that exhibited fungalgrowth. The range was about one to seven fungal organisms per patientwith an average of about 2.9 fungal species per patient. Sixty-threepercent of the cultures included Alternaria, 47 percent includedPenicillium, 33 percent included Cladosporium, 33 percent includedAspergillus, 28 percent included Fusarium, and 20 percent includedCandida.

In a separate study, twelve control individuals (i.e., persons nothaving chronic rhinosinusitis) had mucus samples collected and analyzedas described herein. All twelve (100%) had positive fungal cultures.Specifically, a total of seven different fungal organisms were culturedwith an average of about 2.25 different fungal organisms per person anda range of one to four. Fifty percent of the cultures includedCladosporium, 42 percent included Alternaria, 33 percent includedGeotrichum, 33 percent included Aspergillus, 25 percent includedPenicillium, 8 percent included Acremonium, and 8 percent includedCandida. These results indicate that fungal organisms live thenasal-paranasal mucus of most, if not all, humans.

Example 2 Treating and Preventing Non-Invasive Fungus-InducedRhinosinusitis

One hundred and thirty-two consecutive rhinosinusitis patients wereentered into a study to evaluate the use of an antifungal agent to treatnon-invasive fungus-induced rhinosinusitis. After diagnostic analysis,125 of the 132 patients (95%) had the following criteria: (1) presenceof observable disease within the nasal-paranasal anatomy as evidenced bya CT scan, (2) presence of allergic mucus as evidenced by histologicevaluation of a surgical specimen, and (3) presence of fungal organismswithin nasal-paranasal mucus as evidenced by the ability to culturefungal organisms from a mucus sample. The 125 non-invasivefungus-induced rhinosinusitis patients were started on an antifungaltreatment of about 20 mL of an amphotericin B solution per nostril, twoto four times daily for at least three months. The concentration of theamphotericin B solution was about 100 mg per liter of saline or water. A20 mL bulb was used by the patient to mucoadminister the amphotericin Bsolution into the patient's nasal-paranasal anatomy. Data were compiledfor 53 of the patients who had returned for their three month follow-upanalysis.

In addition to patient interviewing, CT scan analysis, visualexamination, and fungal culture analysis, two types of evaluations wereused to score the success of the treatment: an endoscopic evaluation anda patient symptom evaluation. These evaluations were scored as follows:

Endoscopic Evaluation

Stage 0: no evidence of disease

Stage 1: polypoid changes/polyps seen by endoscopy only

Stage 2: polyps in the middle meatus

Stage 3: polyps filling the nasal cavity

Patient Symptom Evaluation

Stage −2: very bad/much worse

Stage −1: bad/worse

Stage 0: baseline/no change

Stage 1: good/improved

Stage 2: very good/free of symptoms

Endoscopic evaluation revealed that 33 of the 53 patients went fromstage 2 or 3 to stage 0 after three months. Six of these 33 casesshowing no evidence of disease were confirmed by CT scans. For example,one patient having had no recent surgeries and taking no steroids wasdiagnosed with bilateral rhinosinusitis since a CT scan revealedbilateral involvement (FIG. 1). The patient then received the treatmentof 20 mL of an amphotericin B solution (100 mg/L) per nostril two timesdaily. After four months of continuous antifungal treatment, a CT scanwas taken to show the complete disappearance of opacity and symptomscharacteristic of rhinosinusitis (FIG. 2).

Eleven of the 53 patients went from endoscopic evaluation stage 2 or 3to stage 1 after three months. The other nine patients did not respondto the treatment. Five of the nine non-responding patients hadpreviously collected mucus samples that were available for examination.Analysis of these five available samples revealed that all five patientshad fungal organisms within their mucus that were resistant toamphotericin B, the antifungal agent used for the treatment.

Patient symptom evaluation revealed that 44 of the 53 patients gavethemselves a stage 2, three of the 53 gave themselves stage 1, and sixof the 53 gave themselves a stage 0 after treatment. The nine patientsgiving themselves a stage 1 or 0 were the same nine patients that didnot have any response as measured by endoscopic evaluation, five ofwhich were shown to contain fungal organisms resistant to amphotericinB. In a subsequent review of another patient cohort, severalnon-responding patients were found not to contain amphotericinB-resistant fungal organisms.

In addition, several patients had mucus samples collected and analyzedbefore and after antifungal treatment. Comparing results from theevaluation of mucus samples before and after antifungal treatmentrevealed that the number of different fungal species in those patientswas remarkably reduced after antifungal treatment as determined byfungal organism culturing techniques. Thus, the rhinosinusitis patientswere asymptomatic and contained less fungus in their mucus aftertreatment with an antifungal agent.

In a separate unique study, a patient was diagnosed with rhinosinusitisin the left paranasal sinuses since a CT scan showed inflammatorydisease characteristic of rhinosinusitis-related opacification in theleft paranasal sinuses. A RAST assay to Alternaria showed 6.23 kilounits per liter (KU/L) and bi-lateral fungal cultures confirmedAlternaria growth in each nostril. Only the left nasal-paranasal side,however, received surgery as well as intra-operative and post-operativetreatment with about 20 mL of an amphotericin B solution (100 mg/L) twoto four times daily. At every post-operative visit, the patient's leftparanasal sinuses were clear of disease. A RAST reading taken eight toten weeks after the disappearance of rhinosinusitis symptoms in thepatient's left sinuses, however, was 7.16 KU/L. This represents anincrease over the first RAST reading. At six months post-operation, thepatient was diagnosed with rhinosinusitis in the right paranasal sinusesbased on a CT scan and a 10.0 KU/L RAST reading to Alternaria. Aftersurgery on the patient's right paranasal sinuses and antifungaltreatment on both sides using about 20 mL of an amphotericin B solution(100 mg/L) per nostril two to four times daily for about seven weeks,the patient remained symptom-free and had a RAST reading of 4.47 KU/L.Six months after this last surgery, the patient remained symptom-freeand disease-free as evidenced by a CT scan.

Taken together, these results indicate that an appropriate irrigationwith a properly administered antifungal agent to a single side resultedin prevention of inflammatory symptoms on that side. In addition, thepreviously detected fungal load in the initially untreated side (rightside) was sufficient to eventually cause presentation of visual orpalpable rhinosinusitis symptoms in that initially untreated side.Further, the fungal organisms present in the initially untreated side(right side) induced high IgE titers, as shown by IgE readings from theRAST assays, independent of concurrent reduction of fungal organisms byantifungal treatment applied to the left side. In this case, a reductionof IgE readings from the RAST assays was only observed after irrigatingboth sides with an antifungal agent. Thus, the reduction of IgE and theprevention of disease symptoms coincided with the treatment of bothsides with an antifungal agent.

To further evaluate the use of an antifungal agent to treat non-invasivefungus-induced rhinosinusitis, patient information was collected foreach patient returning to the physician's office during a one week timeperiod. Only previously seen patients that were instructed to use theantifungal amphotericin B nasal irrigations were entered into thisstudy.

During a one week period, twenty patients returned to the physician'soffice (Table II). The average age of the returning patients was 47years (range 16-74 years). The patients were using the amphotericin Birrigations for an average duration of about six months (range 1-16months). Some patients had a nasal surgery as recent as one month whileothers never had such a surgery. In addition, some patients were usingtopical and systemic steroid therapy. Further, some patients were usingan antibiotic nasal irrigation in addition to the antifungalirrigations. The antibacterial solution contained 80 mg gentamicin per Lsaline (Wilson's solution). Some patients mixed the antibacterialsolution with the antifungal solution and then performed the nasalirrigations while others used each solution separately in a sequentialmanner. Some patients also had other diseases including asthma (15 ofthe 20 patients) and colitis (2 of the 20 patients).

Upon endoscopic evaluation, most patients had an observable improvementin their non-invasive fungus-induced rhinosinusitis condition. Theseobservable improvements correlated with the symptom improvement scoresgiven by each patient. One patient stopped the nasal amphotericin Birrigations after two months. Eight months later that patient exhibitedrecurrent symptoms of the non-invasive fungus-induced rhinosinusitiscondition. Two other patients switched from an amphotericin B solution(duration: 3 months; frequency: twice a day) to an itraconazole solution(duration: 1 month; frequency: twice a day). One reported feeling betterafter using the itraconazole solution for only seven days. Takentogether, these results indicate that antifungal agents can be usedeffectively to treat non-invasive fungus-induced rhinosinusitis.

TABLE II Patient data collected during a one week period. EndoscopySymptom Duration Last Steroid Other Score Score Age (Ampho) FrequencySurgery Therapy Diseases B A B A 59  3 mon¹ 2 × day  2 mon No Asthma NANA −1   1 40  1 mon 2 × day None Yes⁸ Asthma 2 1 −1 +2 Colitis 63  4 mon1 × day  4 mon No Colitis 1 0 −1 +1 16 12 mon² 1 × day 26 mon TopicalAsthma 2 0 −1 +2 44 12 mon² 2 × day  9 mon No Asthma 3 0 −1 +1 40  2mon³ 2 × day 12 mon No No 3 3 −1   0 23 16 mon 2 × day 10 mon No Asthma3 0 −1 +1 48  4 mon 2 × day >10 yrs Topical No 2 0 −1   0 50  4 mon 2 ×day  4 mon No Asthma 1 0 −1 +1 45  2 mon 2 × day None Topical Asthma 1NA −1   0 74  2 mon 2 × day  >4 yrs Topical No 2 NA −1 +1 57  2 mon 2 ×day  1 mon Systemic Asthma 2 0 −1 +2 Topical 16 12 mon⁴ 1 × day 12 monNo  Asthma¹² 3 0 −1 +2 71  7 mon 2 × day  >6 yrs No Asthma 3 3 −2 −2 3812 mon 2 × day  >6 yrs Yes⁹ Asthma 3 3 −1   0 38  5 mon 2 × day 28 monNo Asthma 0 0 −2 +2 66 13 mon 2 × day  >4 yrs Topical No 3 3 −2   0 70 3 mon⁵ 2 × day  >2 yrs Systemic¹⁰ Asthma 3 3 −2 +1 Topical 32  3 mon⁶ 2× day  5 mon No Asthma 3 1 −2 +2 47  2 mon⁷ 2 × day  >3 yrs Systemic¹¹Asthma 3 0 −2   0 Topical B, before antifungal treatment; A, afterantifungal treatment ¹Also irrigated with Wilson's solution (80 mggentamicin/L saline) twice a day ²Also intermittently irrigated withWilson's solution ³Stopped the nasal irrigations 8 months earlier anddisease has recurred ⁴Also irrigated with Wilson's solution (80 mggentamicin/L saline) once a day ⁵Felt better seven days after switchingfrom amphotericin B (duration: 3 months; frequency: twice a day) toitraconazole (duration: 1 month; frequency: twice a day) nasalirrigations ⁶Switched to itraconazole irrigations (duration: 1 month;frequency: twice a day) after 3 months on amphotericin B ⁷Also irrigatedwith Wilson's solution (duration: 1 year) ⁸Received Kenalog 40 IM MedvalDose Pack 1 month earlier ⁹Received Kenalog Shot 6 months earlier¹⁰Received Prednisone for 1 week ¹¹Received systemic steroid treatmentfor 3 years ¹²Quit taking theophylline and tylade since startingantifungal irrigations

Example 3 Treating and Preventing Non-Invasive Fungus-InducedRhinosinusitis in Patients Without Previous Nasal Surgery

The following three non-invasive fungus-induced rhinosinusitis patientsdid not have a previous nasal surgery.

A 61 year old male was diagnosed with non-invasive fungus-inducedrhinosinusitis and instructed to perform amphotericin B irrigationstwice a day. Before starting the treatment, endoscopic evaluationrevealed polyps filling her nasal cavity (endoscopic score 3) and thepatient gave herself a symptom score of −1. After using the amphotericinB irrigations for fourteen months, endoscopic evaluation revealed noevidence of disease (endoscopic score 0) and the patient gave herself asymptom score of +2.

A 64 year old female was diagnosed with non-invasive fungus-inducedrhinosinusitis and instructed to perform amphotericin B irrigationstwice a day, which was later increased to four times a day. Beforestarting the treatment, endoscopic evaluation revealed evidence ofpolypoid changes (endoscopic score 1) and the patient gave himself asymptom score of −1. After using the amphotericin B irrigations forsixteen months, endoscopic evaluation revealed no evidence of disease(endoscopic score 0) and the patient gave himself a symptom score of +2.

A 54 year old male was diagnosed with non-invasive fungus-inducedrhinosinusitis and instructed to perform amphotericin B irrigationstwice a day. This patient had been given intramuscular steroid shotsevery 3 to 8 months with the last shot being administered about sevenmonths prior to starting the amphotericin B irrigations. Before startingthe antifungal treatment, endoscopic evaluation revealed no evidence ofdisease (endoscopic score 0) but the patient gave herself a symptomscore of −1. After using the amphotericin B irrigations for four months,endoscopic evaluation again revealed no evidence of disease (endoscopicscore 0), however, the patient gave herself a symptom score of +1.

Example 4 Diminishing Eosinophilia Using an Antifungal Treatment

A 67 year old female was diagnosed with non-invasive fungus-inducedrhinosinusitis and instructed to perform amphotericin B irrigationstwice a day. After nine months of amphotericin B irrigations, thepatient underwent sinus surgery for further improvement. During thesurgery mucosal biopsies were collected and the eosinophil countcompared to the those obtained from biopsies collected from the patientduring a surgery prior to the antifungal treatment.

The eosinophil count in all the mucosal biopsies from all the sinuses,except the frontal was diminished (<5%) after antifungal treatment. Theeosinophil count in the frontal sinus biopsy was 10%. In addition,allergic mucus appeared to be present in the frontal sinus, presumablybecause the amphotericin B irrigations did not get to the frontal sinusdue to the frontal sinus obstruction. Thus, the previously observedhypereosinophilia had diminished to normal in all the treated sinusareas.

Example 5 Treating and Preventing Chronic Asthma Symptoms

Thirty-seven of the 53 patients in the study described in Example 2 hadpreviously diagnosed chronic asthma. After three months of antifungaltreatment, 28 of the 37 asthmatic patients upon questioning declared animprovement or complete elimination of asthma symptoms. Four of these 28were analyzed using a pulmonary function test after antifungal treatmentsince they had taken a similar test before antifungal treatment.Comparing the results before and after antifungal treatment confirmedthat all four of these asthma patients had improved pulmonary function.In addition, 26 of the 28 patients no longer exhibiting asthma symptomsstopped taking their asthma medication. Twenty-three of these 26patients were taking systemic steroids for asthma prior to theantifungal treatment, but none have subsequently taken steroids afterstarting the antifungal treatment.

In a separate study, sputum samples from the lung were collected fromseven asthma patients. Culture analysis of these samples revealed thepresence of fungal organisms in each sample. Specifically, Candidaalbicans, Penicillium, Fusarium, Scopulariopsis, Cryptococcus,Cladosporium, Aspergillus, Aspergillus fumigatus, Aspergillus nidulans,and yeast were cultured. The number of different fungal species culturedfrom each sputum sample ranged from one to five.

Example 6 Itraconazole Formulations

Itraconazole formulations were made by dissolving itraconazole intopolyethylene glycol (PEG) to form an itraconazole stock solution. Theitraconazole was obtained from 100 mg itraconazole capsules (JanssenPharmaceutica, Inc.). Typically, PEG 400 was used to dissolve theitraconazole. Once dissolved, the stock solution was filtered to removeany insoluble material. Then, the stock solution was prepared for use bydilution with sterile water.

Specifically, twenty 100 mg itraconazole capsules were opened and thespheres having itraconazole were placed into a graduated cylinder. Oneliter of heated (70° C.) PEG-400 was added to the graduated cylindercontaining the itraconazole. The mixture was then placed onto a stirringhot plate and maintained at 70° C. for 30 minutes. After 30 minutes, thehot suspension was filtered through a urine stone filter into a glasscontainer and allowed to cool to room temperature. Once cooled, 100 mLof the filtered solution was placed into an empty plastic bottle. Then,900 mL of sterile water was added and the solution mixed. Once mixed,one drop of flavoring was added (peppermint oil). This proceduretypically resulted in a solution containing about 98.8 μg to about 111μg of itraconazole per mL.

The following concentrations of itraconazole were determined for eachindicated solution by HPLC (Table III).

TABLE III Concentrations of itraconazole in solution. SolubleItraconazole Solution Concentration (μg/mL) 2000 mg Itraconazole fromcapsules into 1 L 1839  PEG-400 (stock solution) 100 mL stock solutiondiluted with 900 mL sterile 113 water 500 mg Itraconazole powder “AS”into 250 mL 1951  PEG-400 (stock solution) 100 mL stock solution dilutedwith 900 mL sterile  85 water 2000 mg Itraconazole from capsules into 1L PEG-400 (stock solution) 100 mL stock solution plus 150 mL PEG-400 179diluted with 750 mL sterile water 2000 mg Itraconazole from capsulesinto 1 L PEG-400 (stock solution) 100 mL stock solution plus 25 mLPEG-400 155 diluted with 875 mL sterile water

An itraconazole formulation containing a steroid was also made.Specifically, the contents from two PULMICORT 200 μg inhalers (about 91μg of budesonide total) was added to an itraconazole PEG-400 stocksolution at 70° C. for about 15 minutes. The budesonide was added about5 minutes after the itraconazole powder was dissolved into the PEG-400.After cooling to room temperature, some precipitation occurred. Thisinsoluble material was removed by filtering the solution through finefilter paper under vacuum. The filter was dried and the capturedprecipitate was measured (36-40 μg) Thus, about 54 to 50 μg of steroidremained in the solution/fine suspension.

Example 7 Treating and Preventing Non-Invasive Fungus-InducedRhinosinusitis Using Itraconazole

Three non-invasive fungus-induced rhinosinusitis patients (33 year oldmale, 70 year male, and 57 year old female) were instructed to performnasal irrigation with an itraconazole solution. The itraconazolesolution contained about 100 mg of itraconazole per L of solution (10%PEG-400 in sterile water) and was prepared as described herein. Twopatients were instructed to perform itraconazole irrigations becausethey did not respond to amphotericin B irrigations. Each patientreported marked improvement in symptoms within two weeks of starting theitraconazole irrigations (symptom scores: −1 to +2 and −1 to +1).Sixteen days after starting the itraconazole irrigations, one of thesetwo patients exhibited improvement as revealed by endoscopic analysis(endoscopic score: from 1 to 0 for right side and from 1 to 1 for leftside). In addition, this patient indicated that her asthma symptoms haddramatically improved and she reduced her asthma medication (Flovent andServent) from twice a day to once a day.

The third patient was instructed to perform itraconazole irrigationsbecause of an adverse local reaction to amphotericin B (burningsensation). After treatment with itraconazole, that patient reportedsymptom improvement (symptom score: from −1 to 0). In addition, thatpatient did not have any adverse local reaction or problems with theitraconazole irrigations.

Example 8 Treating and Preventing Chronic Asthma Symptoms UsingItraconazole

A 32 year old white male patient with no history or symptoms of chronicrhinosinusitis exhibited significant asthma symptoms despite medicaltherapy with systemic and topical steroids and frequent use of abronchodilator. Sputum and nasal-paranasal mucus samples were collectedand analyzed. Culture analysis revealed the presence of Candida albicansin the sputum and Penicillium, Geotrichum, Alternaria, and Cladosporiumspecies in the nasal-paranasal mucus.

The patient was started on an antifungal treatment of about 20 mL of anitraconazole solution per nostril, two times daily. The concentration ofthe itraconazole solution was about 100 mg per liter. After a few weeks,the patient took his last course of systemic steroids. About two monthsafter stopping the systemic steroids, the patient also stopped using thetopical steroids as well as the bronchodilator. Since stopping allsteroid therapy, the patient's symptoms improved dramatically.Specifically, the patient reports no episodes of shortness of breath andno wheezing during the four to five month period since stopping allsteroid therapy.

Objective analysis also revealed dramatic improvement. In a studyconducted before antifungal treatment, the patient exhibited abnormalpulmonary function. After seven months of continuous antifungalirrigations as described with the later four to five months being freeof all steroid therapy, the patient exhibited improved pulmonaryfunction. Specifically, the forced vital capacity (FVC) of the lungimproved from 3.99 liter before treatment to 4.80 liter after treatment,an increase of 20.30%; the forced expiratory volume in 1 second (FEV1),a marker for the degree of lower airway resistance, improved from 3.34liter before treatment to 4.27 liter after treatment, an increase of27.84%; the maximal forced expiratory flow (FEFmax) improved from 9.1liter per second before treatment to 12.6 liter per second aftertreatment, an increase of 38.46%; and the maximum voluntary ventilation(MVV) improved from 119 liter per minute before treatment to 156 literper minute after treatment, an improve of 31.90%.

In summary, the objective markers revealed an improvement of thepulmonary function between 20.3% and 38.46%, despite no medical therapyother than the antifungal nasal irrigations during the previous four tofive months. These results indicate that chronic asthma symptoms can betreated and prevented by mucoadministering antifungal agents to theairways.

Sometime after this patient's asthma symptoms improved, the patientstopped using the itraconazole irrigations. After four to six weeks ofnot using the itraconazole irrigations, the patient's asthma symptomsreturned. At that time, the patient inhaled steroids only to control theasthma symptoms. After about four to six weeks, the patient switchedfrom using the steroid inhaler to using an itraconazole powder inhaler.Specifically, the patient was instructed to inhale about 400 μg of pureitraconazole per day using a powdered inhaler. The itraconazole powderwas an authentic substance “AS” (Janssen Pharmaceutica, Inc.). Theinhaler was a Pulmicort 200 μg TURBOHALER® manufactured by ASTRApharmaceuticals. This inhaled was designed for metered doses ofbudesonide inhalation powder, but was adapted to administeritraconazole. The patient has been asymptomatic for four weeks, andcontinues to use the itraconazole powdered treatment. The patient alsohad at least one nasal polyp initially. That polyp was noticeablyreduced in viability by the second week of treatment.

Another asthma patient also was instructed to inhale about 200 μg ofpure itraconazole per day using an adapted Pulmicort 200 μg TURBOHALER®.After about 2 weeks, the patient's condition was markedly improved. Thepatient remains on the treatment.

An asthma patient having non-invasive fungus-induced rhinosinusitis wastreated with itraconazole using a nebulizer. Specifically, about two mLof an itraconazole solution (about 10 mg itraconazole per mL PEG-400)was applied per day in a nebulizer. The nebulizer was an air pressurizedPULMO-MATE brand manufactured by DeVillbis. After about two weeks, thepatient demonstrated improvement in both asthma and non-invasivefungus-induced rhinosinusitis conditions as evidenced by an overallimprovement of symptom scores. Improvements were also noted in one weekincrements.

Example 9 Identifying Non-Invasive Fungus-Induced Otitis Media

Mucus samples from the middle ear were collected using a suction trapfrom three patients diagnosed with chronic otitis media. Cultureanalysis of the samples revealed the presence of fungal organisms.Specifically, the mucus sample from the first patient was positive forCandida and Trichophyton rubrum species, the mucus sample from thesecond patient was positive for Penicillium species, and the mucussample from the third patient was positive for Aspergillus species. Inaddition, microscopic examination revealed a large number ofdegenerating eosinophils within each mucus sample. Thus, these resultsindicate that chronic otitis media is most likely caused by non-invasivefungal organisms. Moreover, it appears that chronic otitis media is anon-invasive fungus-induced mucositis that can be treated and preventedusing the antifungal treatment and prevention approaches describedherein.

Example 10 Treating Non-Invasive Fungus-Induced Intestinal Mucositis

Three out of five consecutive patients with chronic rhinosinusitisreported having a history of colitis. Two patients were started on anantifungal treatment of one capsule of itraconazole (provided by JanssenPharmaceutica, Inc.) per day. The capsule contained 100 mg ofitraconazole. Each patient was instructed to take the capsule beforebedtime a minimum of two hours after their last meal and without anycola. Food and cola beverages increase absorption of the drug. Whentaken as described, about 50 percent of the itraconazole should remainin the bowel lumen for treatment of non-invasive fungus-inducedintestinal mucositis symptoms.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a mammal havingnon-invasive fungus-induced intestinal mucositis, comprisingmucoadministering to the digestive tract of said mammal a formulation inan amount, at a frequency, and for a duration effective to reduce oreliminate said non-invasive fungus-induced intestinal mucositis, saidformulation comprising an antifungal agent, wherein saidmucoadministration comprises orally applying said formulation to saiddigestive tract, and wherein said duration is greater than about 30days.
 2. The method of claim 1, wherein said mammal is a human.
 3. Themethod of claim 1, wherein said mammal is nonatopic.
 4. The method ofclaim 1, wherein said mammal is immunocompetent.
 5. The method of claim1, wherein said non-invasive fungus-induced intestinal mucositis ischaracterized by polyp formation or polypoid change.
 6. The method ofclaim 1, wherein said non-invasive fungus-induced intestinal mucositisis chronic.
 7. The method of claim 1, wherein said formulation is in asolid form.
 8. The method of claim 1, wherein said formulation is in theform of a capsule.
 9. The method of claim 8, wherein said capsule is aregulated release capsule.
 10. The method of claim 9, wherein saidregulated release capsule is a pH regulated release capsule.
 11. Themethod of claim 9, wherein said regulated release capsule is a timeregulated release capsule.
 12. The method of claim 1, wherein saidmucoadministration is a direct mucoadministration.
 13. The method ofclaim 1, wherein said antifungal agent comprises a macrolide.
 14. Themethod of claim 1, wherein said antifungal agent comprises an azole. 15.The method of claim 1, wherein said antifungal agent interpolates fungalcell wall components.
 16. The method of claim 1, wherein said antifungalagent comprises a sterol inhibitor.
 17. The method of claim 1, whereinsaid antifungal agent comprises an antifungal agent selected from thegroup consisting of amphotericin B, ketoconazole, itraconazole,saperconazole, voriconazole, flucytosine, miconazole, fluconazole,griseofulvin, clotrimazole, econazole, terconazole, butoconazole,oxiconazole, sulconazole, ciclopirox olamine, haloprogin, tolnaftate,naftifine, terbinafine hydrochloride, morpholines, nystatin, natamycin,butenafine, undecylenic acid, Whitefield's ointment, propionic acid, andcaprylic acid.
 18. The method of claim 1, wherein said antifungal agentcomprises an antifungal agent selected from the group consisting ofketoconazole, itraconazole, saperconazole, and voriconazole.
 19. Themethod of claim 1, wherein said antifungal agent comprises amphotericinB.
 20. The method of claim 1, wherein said antifungal agent comprisesitraconazole.
 21. The method of claim 1, wherein said formulationcomprises about 0.01 ng to about 1000 mg of said antifungal agent. 22.The method of claim 1, wherein said formulation comprises about 1 ng toabout 500 mg of said antifungal agent.
 23. The method of claim 1,wherein said formulation comprises about 100 mg of said antifungalagent.
 24. The method of claim 1, wherein said formulation comprises aplurality of antifungal agents.
 25. The method of claim 1, wherein saideffective amount of said formulation comprises about 0.01 ng to about1000 mg of said antifungal agent per kg of body weight of said mammal.26. The method of claim 1, wherein said effective amount of saidformulation comprises about 1 ng to about 500 mg of said antifungalagent per kg of body weight of said mammal.
 27. The method of claim 1,wherein said effective amount of said formulation remains constantduring said effective duration.
 28. The method of claim 1, wherein saideffective frequency of said mucoadministration is from about four timesa day to about once every other week.
 29. The method of claim 1, whereinsaid effective frequency of said mucoadministration is from about twicea day to about once a week.
 30. The method of claim 1, wherein saideffective frequency of said mucoadministration is more frequent thanonce a day.
 31. The method of claim 1, wherein said effective frequencyof said mucoadministration is more frequent than once a week.
 32. Themethod of claim 1, wherein said effective duration is greater than about60 days.
 33. The method of claim 1, wherein said effective duration isgreater than about 90 days.
 34. The method of claim 1, wherein saidformulation comprises a compound selected from the group consisting ofpharmaceutically acceptable aqueous vehicles, pharmaceuticallyacceptable solid vehicles, mucolytic agents, antibacterial agents,anti-inflammatory agents, immunosuppressants, dilators,vaso-constrictors, steroids, and therapeutic compounds.
 35. The methodof claim 1, wherein said method comprises administering to said mammal asecond formulation.
 36. The method of claim 35, wherein said secondformulation comprises a compound selected from the group consisting ofantifungal agents, pharmaceutically acceptable aqueous vehicles,pharmaceutically acceptable solid vehicles, mucolytic agents,antibacterial agents, anti-inflammatory agents, immunosuppressants,dilators, vaso-constrictors, steroids, and therapeutic compounds. 37.The method of claim 1, said method comprising, after saidmucoadministration, prophylactically mucoadministering to said mammal aprophylactic formulation in an amount, at a frequency, and for aduration effective to prevent said non-invasive fungus-inducedintestinal mucositis, said prophylactic formulation comprising anantifungal agent.
 38. The method of claim 37, wherein said prophylacticmucoadministration comprises direct mucoadministration.
 39. A method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced intestinal mucositis, comprising mucoadministering tosaid mammal a formulation in an amount, at a frequency, and for aduration effective to prevent said non-invasive fungus-inducedintestinal mucositis, said formulation comprising an antifungal agent.40. A method for treating a mammal having a non-invasive fungus-inducedintestinal mucositis, comprising the steps of: a) identifying saidmammal, and b) mucoadministering to at least a portion of the digestivetract of said mammal a formulation in an amount, at a frequency, and fora duration effective to reduce or eliminate said non-invasivefungus-induced intestinal mucositis, said formulation comprising anantifungal agent, wherein said mucoadministration comprises orallyapplying said formulation to said digestive tract, and wherein saidduration is greater than about 30 days.
 41. A method forprophylactically treating a mammal at risk for developing non-invasivefungus-induced intestinal mucositis, comprising the steps of: a)identifying said mammal, and b) mucoadministering to at least a portionof the digestive tract of said mammal a formulation in an amount, at afrequency, and for a duration effective to prevent said non-invasivefungus-induced intestinal mucositis, said formulation comprising anantifungal agent.
 42. An article of manufacture, comprising packagingmaterial and a formulation contained within said packaging material,wherein said formulation comprises an antifungal agent and wherein saidpackaging material comprises a label or package insert indicating thatsaid formulation can be mucoadministered to a mammal having non-invasivefungus-induced intestinal mucositis in an amount, at a frequency, andfor a duration effective to reduce or eliminate said non-invasivefungus-induced intestinal mucositis.
 43. An article of manufacture,comprising packaging material and a formulation contained within saidpackaging material, wherein said formulation comprises an antifungalagent and wherein said packaging material comprises a label or packageinsert indicating that said formulation can be mucoadministered to amammal at risk for developing non-invasive fungus-induced intestinalmucositis in an amount, at a frequency, and for a duration effective toprevent said non-invasive fungus-induced intestinal mucositis.
 44. Amethod for treating a human having non-invasive fungus-inducedintestinal mucositis, comprising mucoadministering to the digestivetract of said human a formulation in an amount, at a frequency, and fora duration effective to reduce or eliminate said non-invasivefungus-induced intestinal mucositis, said formulation comprising anantifungal agent, wherein said mucoadministration comprises orallyapplying said formulation to said digestive tract, and wherein saidfrequency is from about twice a day to about once a week.
 45. The methodof claim 44, wherein said human is immunocompetent.
 46. The method ofclaim 44, wherein said non-invasive fungus-induced intestinal mucositisis characterized by polyp formation or polypoid change.
 47. The methodof claim 44, wherein said formulation is in the form of a capsule. 48.The method of claim 47, wherein said capsule is a regulated releasecapsule.
 49. The method of claim 48, wherein said regulated releasecapsule is a pH regulated release capsule.
 50. The method of claim 48,wherein said regulated release capsule is a time regulated releasecapsule.
 51. The method of claim 44, wherein said mucoadministration isa direct mucoadministration.
 52. The method of claim 44, wherein saidantifungal agent comprises an azole.
 53. The method of claim 44, whereinsaid antifungal agent comprises an antifungal agent selected from thegroup consisting of ketoconazole, itraconazole, saperconazole,voriconazole, flucytosine, miconazole, fluconazole, griseofulvin,clotrimazole, econazole, terconazole, butoconazole, oxiconazole,sulconazole, ciclopirox olamine, haloprogin, tolnaftate, naftifine,terbinafine hydrochloride, morpholines, nystatin, natamycin, butenafine,undecylenic acid, Whitefield's ointment, propionic acid, and caprylicacid.
 54. The method of claim 44, wherein said antifungal agentcomprises an antifungal agent selected from the group consisting ofketoconazole, itraconazole, saperconazole, and voriconazole.
 55. Themethod of claim 44, wherein said antifungal agent comprises amphotericinB.
 56. The method of claim 44, wherein said antifungal agent comprisesitraconazole.
 57. The method of claim 44, wherein said formulationcomprises about 0.01 ng to about 1000 mg of said antifungal agent. 58.The method of claim 44, wherein said formulation comprises a pluralityof antifungal agents.
 59. The method of claim 44, wherein said effectiveamount of said formulation comprises about 0.01 ng to about 1000 mg ofsaid antifungal agent per kg of body weight of said human.
 60. Themethod of claim 44, wherein said effective amount of said formulationcomprises about 1 ng to about 500 mg of said antifungal agent per kg ofbody weight of said human.
 61. The method of claim 44, wherein saideffective amount of said formulation remains constant during saideffective duration.
 62. The method of claim 44, wherein said effectiveduration is greater than about 7 days.
 63. The method of claim 44,wherein said effective duration is greater than about 14 days.
 64. Themethod of claim 44, wherein said effective duration is greater thanabout 30 days.
 65. The method of claim 44, wherein said effectiveduration is greater than about 60 days.
 66. The method of claim 44,wherein said effective duration is greater than about 90 days.
 67. Themethod of claim 44, wherein said formulation comprises a compoundselected from the group consisting of pharmaceutically acceptableaqueous vehicles, pharmaceutically acceptable solid vehicles, mucolyticagents, antibacterial agents, anti-inflammatory agents,immunosuppressants, dilators, vaso-constrictors, steroids, andtherapeutic compounds.
 68. The method of claim 44, wherein said methodcomprises administering to said human a second formulation.
 69. Themethod of claim 68, wherein said second formulation comprises a compoundselected from the group consisting of antifungal agents,pharmaceutically acceptable aqueous vehicles, pharmaceuticallyacceptable solid vehicles, mucolytic agents, antibacterial agents,anti-inflammatory agents, immunosuppressants, dilators,vaso-constrictors, steroids, and therapeutic compounds.
 70. The methodof claim 44, said method comprising, after said mucoadministration,prophylactically mucoadministeting to said human a prophylacticformulation in an amount, at a frequency, and for a duration effectiveto prevent said non-invasive fungus-induced intestinal mucositis, saidprophylactic formulation comprising an antifungal agent.
 71. The methodof claim 70, wherein said prophylactic mucoadministration comprisesdirect mucoadministration.